From: Subject: Genome Projects Date: Mon, 16 Feb 2004 13:17:21 -0600 MIME-Version: 1.0 Content-Type: text/html; charset="Windows-1252" Content-Transfer-Encoding: quoted-printable Content-Location: http://www.briefhistoryoflife.com/BHL15GENPROJ.htm X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2800.1165 Genome = Projects

GENOME PROJECTS

Genetic=20 engineeringf

What's a Genome?

A major=20 function of organisms and cells is reproduction. Reproduction is the = duplication=20 (not necessarily exactly) of an organism. Each organism must maintain = the=20 information necessary to reproduce itself. This information is called = the genome=20 of the organism.

The human=20 genome is the genetic information that resides in the cells of the human = body.=20 Most of this information is stored in 23 pairs of chromosomes in the = cell=20 nucleus. The chromosomes = are long=20 string-like molecules which are made of units called nucleic acids. There are about three billion = pairs of=20 nucleic acid units that code for tens of thousands of individual = genes.

The Human=20 Genome Project is a variety of research efforts aimed at understanding = the=20 structure and function of human genetics. =20 A draft of the 3 billion base pair human genome has been = completed, but=20 much remains to be done. = Genome=20 related projects are working to improve the tools for analyzing genes, = proteins,=20 and biological systems. = Other=20 projects are exploring variations between different human genomes; and = studying=20 the genomes of other organisms. =20 Additional areas of study include feedback and control = mechanisms,=20 intercellular communication and cooperation, and even the study of the = molecular=20 basis of learning and consciousness. WHY? WHAT DO THEY HOPE TO=20 GAIN?

Who is paying for this?

Life=20 science is supported by the public (governments), by industries and=20 corporations, by schools and universities, and by private foundations = and=20 individuals. Billions of dollars are being spent on the Human Genome and = related=20 molecular biology topics. = Drug=20 companies and other private corporations are expending large sums in = hope of=20 reaping rewards. = Techniques, gene=20 sequences and proteins are being patented at an ever increasing=20 rate.

It is=20 likely that important experiments will be prevented by uninformed public = fears.=20 Conversely dangerous activities may well be permitted. The publics = legislating=20 the permissibility of these applications and providing the moneys for = this=20 research need to make informed decisions. =20 Policies regarding the ownership of genetic knowledge must = balance the=20 rights of people, organizations, and mankind to patent and otherwise = control=20 this knowledge.

In today's laboratories, new = types of=20 organisms are routinely created by genetic engineering technology. = Transgenic=20 mice have been developed which contain genetic material from humans and = other=20 species. Today enzymes, hormones, and chemicals such as insulin and = human growth=20 hormones are routinely being produced by genetically engineered = bacteria. Cells=20 can be grown in test tubes outside of the plants and animals from which = they=20 came. It is possible to modify the characteristics of plants and animals = to meet=20 our needs using genetic engineering technology. An example is introduction of = genes to=20 produce pesticides in food plants. =20 Genetically engineered cells are being inoculated into humans to = cure or=20 combat diseases such as cancer and diabetes. Man made viruses are being = used to carry=20 genetic material into the cells of people.

Extensive=20 debate has arisen over the release of genetically engineered species = into the=20 environment. This debate will intensify as the potential increases to=20 genetically engineer our foods, our medicines, and our children.=20

Techniques=20 have been developed to prepare exact copies of higher organisms. Many species including mice, = sheep, and=20 probably humans have already been cloned. =20 What limits should be placed on preparation of cloned human = cells,=20 tissues, and even whole human beings? =20 How can these limits be enforced?

Tests for=20 genetic characteristics are being developed and increasingly used. What rights do individuals = have to their=20 own genetic information? = Can others=20 patent one of your genes? = How can=20 genetic discrimination by employers, insurers, and others be=20 prevented?

Concepts:

HUGO - history, alta, gilbert, dulbecco, big = science,=20 players, 5 year plans

Junk DNA

Genes CF Huntington=92s disease, alcoholism, = perfect pitch,=20 cancer

Model organisms

e. coli, yeast, worm, plants, mouse other

Structure, Program, Status

WHAT IS THE HUMAN GENOME PROJECT

The goal of the human genome project is to = determine the=20 location and molecular details of the human genome. The 3 billion base pairs of = DNA in the=20 human chromosomes that contains the million or so genes of humans. Most accounts say that there = are about=20 100,000 genes in the human genome. =20 Earlier it was believed that most (over 90 perhaps 97%) of the = human=20 genome was "junk DNA".

Junk DNA

When I attended the first Human Genome Conference = in 1989,=20 there was criticism of the idea of totally sequencing the human genome = since=20 "97% is junk, it doesn't code for any protein". In the years since then, it = has been=20 discovered that this "junk" DNA plays a vital role in evolution, = regulation,=20 repair, and disease.

The genome, rather than being a catalog of 100,000 = or so=20 genes interspersed with junk, is now viewed as a complex "information = organelle"=20 full of control and maintenance subsytems.

In prokaryotes the genome is almost entirely made = up of genes=20 that code for particular proteins, with some transfer and messenger RNA = genes=20 added.

Many eukaryote genes have regulatory sequences = which are=20 short segments of DNA that are targets for the transcription factors = that=20 activate genes. There may = be as=20 many as five times as many regulatory sequences as there are protein = coding=20 genes in the human genome. = Mutation=20 in a highly repetitive minisatellite may be implicated in many types of=20 cancers. Theodore = Krontiris at=20 Tufts suggests that the mutation activates the nearby ras gene which = encodes a=20 protein important to growth regulation.

In the center and at the ends (telemeres) of = chromosomes is=20 long stretches of satellite DNA which appears to bind proteins that stop = the=20 ends of chromosomes from fraying or breaking apart.

Introns are non-coding regions within the DNA that = codes for=20 a particular protein. = They are=20 removed before the messenger RNA is completed. Leroy Hood at the University = of=20 Washington has shown that some introns are accurately conserved between=20 species. John Mattick = suggests that=20 they take part in regulating gene expression.

Prokaryotes seem to be limited to about 8,000 = different genes=20 whereas eukaryotes may have hundreds of thousand. The activity of prokaryote = genes is=20 regulated by proteins encoded by the genes. This feedback system also = exists in=20 eukaryotes. Introns may = represent a=20 second type of gene regulation system found only in eukaryotes.

Most evolutionary theorists believe that introns = are the=20 remnants of primitive RNA life forms that became incorporated into cells = and=20 evolved with them.

In prokaryotes DNA is translated into RNA which is=20 immediately translated into protein. =20 This all occurs within the cell in the same compartment. Removal of nontranslating = introns might=20 be difficult to accomplish in prokaryotes resulting in nonfunctional=20 proteins. In eukaryotic = cells=20 transcription takes place in the nucleus and translation beyond the = nuclear=20 membrane in the cytoplasm. Mattick suggests that genes might be = regulated by=20 intron encoded RNA that binds either to DNA or RNA.

The XIST gene which shuts down one of the two X = chromosomes=20 in female mammalian cells performs this function without making any=20 protein. It appears that = this gene=20 codes for RNA that binds to the DNA preventing further activity. Another example is that = mutants of 3'=20 untranslated regions of genes in worms, fruit flies, and vertebrates can = suppress the activity of these genes by suppressing translation or by = hastening=20 degradation of their messenger RNAs.

A current catalog of "junk" DNA would include:

Introns - sequences within protein coding genes = that do not=20 code for protein.

Small nucleolar RNAs are encoded by some introns = and may play=20 a part in ribosome assembly.

Satellites are short DNA sequences repeated = hundreds or=20 thousands of times at the center and ends of chromosomes. They may play a role in = maintaining=20 chromosomal structural integrity.

Minisatellites and microsatellites are repetitive = sequences=20 that are found throughout the genome. =20 Defects in these repetitive sequences may be associated with = diseases and=20 cancer.

3' untranslated regions occur after the final = protein coding=20 regions of genes and may contain sequences that regulate gene = activity.

Heterogeneous nuclear RNAs are sequences translated = from DNA=20 some of which code for genes but the function of most is unknown.

Short interspersed elements (SINEs) are repetitive = sequences=20 such as the 300 pair ALU sequence which occurs half a million times in = the human=20 genome. SINEs seem to hop about the genome and may disrupt genes causing = disease=20 such as elephant man's disease.

Long interspersed elements up to 7,000 base = pairs.

Pseudogenes are sequences that would code for = proteins but do=20 not include introns and are not usually expressed in eucaryotes.

The discoveries in molecular biology in the 1970's = led to the=20 genome project. = Restriction enzymes=20 were discovered that would surgically dissect DNA. Ligases that combined DNA led = to genetic=20 engineering and the transfer of genes to bacteria and other organisms = creating=20 biological factories for such molecules as hormones.

In hindsight the first call to sequence the human = genome was=20 from Victor McKusik (Mendelian Inheritance in Man).

In 1984 at a conference in Alta Utah, the idea of = sequencing=20 the human genome was suggested. = In=20 1986 Dulbecco wrote an attention getting article in Science. DOE was looking for = non-military=20 projects and got interested. =

Walter Gilbert at Harvard proclaimed that = "sequencing the=20 human genome is like pursuing the holy grail".

Jim Watson suggested that the project be centered = at=20 NIH. The NAS urged = Congress to=20 begin the HGP. In 1988 = the OTA=20 prepared a report informing Congress of the options.

Genetic linkage maps. =20 Physical maps. = McKusicks=20 book.

By 1990 sequencers handled 10,000 bases per day at = a cost of=20 about $5 per base sequenced. = Today=20 that figure has more than doubled and the cost is below $1 per base.

Manhattan project

Apollo project

Weapons projects =20 Billions for atomic submarines, and even more than the cost of=20 construction for disposal.

In 1970 if you wanted to understand the gene for = hemoglobin,=20 you might want 50 milligram (1/1,000th of an ounce) for analysis. But since the gene is only one = of a=20 million or so, this would represent perhaps 1,500 base pairs in the=20 3,000,000,000 base pairs of the human genome. If you started with raw human = DNA and=20 could separate out the gene without loss you would need 3,000,000,000 / = 1,500 =3D=20 2,000,000 which divided by 1,000 still leaves two thousand ounces or = over 100=20 pounds of human DNA. This = would=20 entail perhaps 50,000 pounds of human cells.

The Human genome project is not really a big = science project=20 in the sense that the Manhattan Project or the Superconducting Super = Collider or=20 the Hubble telescope are big science projects for several reasons.

First the project is not that big. The total proposed effort is = on the=20 order of 3 billion dollars over 15 years. =20

Second the project is not to be performed by one=20 organization. There will = be many=20 groups contributing to the effort. =20

Third the project is really a collection of = different=20 projects. These projects = are:

1. Establishment and maintenance of databases = containing=20 information about nucleic acid and protein sequences from various = different=20 organisms.

2. Creation of maps of human chromasomes. Maps are DNA markers that = would permit=20 scientists to derive the rough location of a particular gene = rapidly.

3. Create repositories of research materials such = as sets of=20 DNA fragments which fully represent the DNA in the human = chromosomes.

4. Development of instruments and technology to = analyze DNA,=20 proteins, structures, etc.

5. Non-human DNA libraries and resources, and

6. Actual sequencing of all or part of the human = genome and=20 that of other organisms.

WHO ARE THE PLAYERS?

NIH spends about 6 billion per year on biomedical=20 research. Of this about = 20 million=20 is for genome work.

DOE spends 230 million in biological research of = which about=20 15 million is for genome work. = DOE=20 rationalizes their entry into the field due to work on biological = effects of=20 radiation. DOE has worked = on=20 development of much of the new equipment for genome work.

NSF is currently a negligible player but is a = logical=20 organization to support technology development.

HHMI is a private organization spending 20 million = per year=20 on genetics and 2 million on data base work.

Foreign countries have devoted rather small amounts = to human=20 genome work although work is being done in France, England, and = Japan.

The genome project will require significant = innovation in the=20 ability to manipulate DNA. = Major=20 advances in analytic instrumentation would be required for cost = effective=20 ordering and sequencing of segments. =20 Theoretical developments in computer science and mathematical = biology and=20 great expansion in the ability to store and manipulate the information = and=20 interface it with other large and diverse genetic databases would be=20 required.

THE HUMAN GENOME

The human genome is made of forty-six chromosomes, = long=20 strands of DNA. Each = strand of DNA=20 is made of subunits called nucleotides. =20 There are three billion pair of nucleotides in the human = genome. Twenty three chromosomes are = inherited=20 from each parent. The = amount of=20 information carried by these chromosomes is immense. Each nucleotide position can = be any of=20 four different nucleotides. = In=20 computer terms this would be two bits of information. Today's best computer chips = hold ten=20 million bits of information on a chip one millimeter by one millimeter = by .00001=20 millimeters. This is = twenty=20 thousand times the volume of the nucleus of a human cell so the computer = chip=20 stores five hundred bits of information in the same space as the nucleus = stores=20 the human genome. The = human genome,=20 by contrast, stores six billion bits of information or more than ten = million=20 times as much information in the same area.

Three nucleotides code for a single amino = acid. The average protein might be = about 500=20 amino acids long. This = would mean=20 that the human genome could code for two million different = proteins. In actuality the genome codes = for only=20 about 60,000.

Genes make up an organism's genotype. Its appearance, function and = behavior=20 make up its phenotype. = Genes and=20 the environment work together to shape the phenotype.

There is no such thing as the single definitive = "human=20 genome". Every gene in = the human=20 gene pool comes in multiple forms perhaps differing in only a single = nucleotide,=20 called alleles. Some = alleles are=20 for rather different characteristics such as the blood type genes. Some individuals have the = 'type A'=20 allele, others the 'type B', others are heterozygous with blood type = 'AB' and=20 some 'type O' have neither. = The=20 gene pool of the cheetah is extremely small. This has been shown by Steve = O'Brian at=20 NIH who found that skin grafts between unrelated cheetahs lasted for = many weeks=20 and often were not rejected at all. =20 This is in contrast to grafts between other cats (or other = species) which=20 are normally rejected in a matter of days to a couple weeks. I am impressed with the work = done on=20 saving the Condor. = Researchers in=20 San Diego evaluated the genome of available condors to select mates with = genetic=20 diversity in breeding condors. = Many=20 killi fish fanciers are concerned about the possible loss of species due = to the=20 destruction of the rain forests and other killifish habitats. We have begun to explore how = many=20 individuals must be kept to maintain a reasonable genetic diversity.

While thinking about cheetahs, the breeding program = at the=20 Wildlife Safari in Oregon was devastated by an infection of feline = peritonitis=20 (which is common among house cats). =20 Almost half of the cheetahs died from the disease whereas nearby = lions=20 were barely affected. It = appears=20 that cheetahs in the wild are solitary and unlikely to encounter the=20 disease. Resistance was = not present=20 in the limited cheetah genome. =20 However cheetahs in zoos are likely to encounter the disease and = it can=20 be devastating. The = behavior of=20 humans, in transferring and introducing organisms from place to place = and=20 increasing communication are likely to increase the danger of harmful = diseases=20 being introduced and spread beyond their natural containment. Another example is = malaria. The benign tertian malaria is = almost=20 nonexistent in equatorial Africa because almost all natives are=20 "Duffy-negative". The = Duffy antigen=20 is a protein-carbohydrate complex on red blood cells that is necessary = for the=20 malaria parasite to attack humans. =20 In fact malaria was practically driven out of the tropics by the=20 resistance of the population. =

Structure function

SHOULD $3 BILLION BE DEVOTED TO THE HUMAN GENOME = PROJECT?

One critic has said that sequencing the complete = human genome=20 is like translating Shakespeare into Sanscrit.

This is based on the fact that knowing the sequence = of the 3=20 billion nucleotides that make up the human genome doesn't tell us = anything about=20 the proteins they code for, the mechanisms of enhancement and = repression,=20 etc.

One of the biggest problems facing molecular = biologists is=20 the problem of determining the structure of a protein from a description = of its=20 sequence.

Daniel Koshland in a Science editorial says failure = to=20 sequence the human genome is "the equivalent of providing iron lungs to = polio=20 victims at the expense of working on a vaccine".

Commercial implications include availability of = data. Should human genome sequences = be subject=20 to copyright?

While ethical and social questions presented by = human genome=20 mapping are similar to those addressed in other settings such as genetic = counseling, the scale "the complete biological book on humankind" is = much=20 greater. Its like saying = we've=20 always had the problem of weapons, so what's different about the atom = bomb?

As mapping work proceeds large family histories = will be=20 studied. Confidentiality = problems=20 exist. Should = confidentiality be=20 broken when the probability is high that serious, avoidable harm would = otherwise=20 come to identifiable individuals?

What about people with HLA-B-27? This is a human leukocyte = antigen that=20 is associated with the disease ankylosing spondylitis. However only one in a thousand = people=20 with the marker get the disease.

Currently genetic counseling and screening is = limited to such=20 factors as Down's syndrome and a few major genetic diseases. Are there limits on the traits = that=20 parents should be able to decide there children must have?

MOUSE MODELS FOR AIDS

In 1942 Avery combined two viruses in the same = cell. Some of them exchanged = coats. This is known as = pseudotyping.

Mice have been developed with a severe combined=20 immunodeficiency (SCID). = These mice=20 have a genetic defect that has wiped out the mouse's own immune = system. These animals are then grafted = with a=20 human immune system. SCID = mice are=20 used in AIDS studies. = They are=20 infected with HIV-1 (Human Immunodeficiency Virus 1). This permits researchers to = determine=20 what happens with the AIDS virus in human cells in living animals. This is more useful than = studies in=20 cultured cells only. An = example of=20 the use of these models is the finding that AIDS virus infected mice = respond to=20 the antiviral drug AZT. = This means=20 that the mouse model may be of use in AIDS drug evaluation.

Robert Gallo at NIH has reported that the AIDS = virus can=20 react with a common mouse virus when they are both in the same = cell. It appears that AIDS viruses = coated with=20 Murine Leukemia Virus coats have been produced. These new viruses have some = disturbing=20 characteristics. First = they=20 reproduce much faster than the AIDS virus. =20 Second they are able to infect types of cells that are immune to = the AIDS=20 virus.

The possibility exists of producing viruses that = can spread=20 by different routes such as being transmitted throught the air.

HUMAN GENE TRANSFER TEST

In 1988 Dr. Stephen Rosenberg of the National = Cancer=20 Institute began an experiment involving injection of genetically = modified=20 tumor-infiltrating lymphocytes (TIL cells) into human patients.

Rosenberg has had success in treating melanomas = with TIL=20 cells that have been cultured with interleukin-2 a growth factor that = stimulates=20 the immune system. This = only works=20 in a fraction of the patients. =20 Why? The gene = transfer=20 experiment is an attempt to develop a better understanding of what is=20 happening. Neomycin = resistance can=20 be easily traced since it is not normal to human cells.

In the gene transfer experiment, the patient's own=20 lymphocytes were extracted and labeled with a bacterial "marker" gene=20 (resistance to the antibiotic neomycin). =20 A retrovirus containing the marker was used as the delivery = system. Then the cells were cultured = to produce=20 a large quantity of cells which were reintroduced into the patient's=20 bloodstream.

Results reported to date are: that the gene labeled = TIL cells=20 did go to the tumor; that the cells survived in the body; and that = adding the=20 marker did not change their characteristics.

If data on the initial five patients continue to = suggest a=20 connection between TIL cells homing in on tumors and tumor shrinkage, = the next=20 step is to identify which of the multiple types of cells in the TIL cell = preparation are doing the job.

In the future it might be possible to use TIL cells = as a=20 mechanism to get anticancer agents into a tumor. The neomycin resistance marker = doesn=92t=20 have any therapeutic effect but it might be possible to use cells with = an=20 anticancer agent. For = example the=20 gene for human tumor necrosis factor has been transferred to human cells = in=20 culture.

The gene transfer experiment was reviewed a total = of 15 times=20 before receiving final approval. = In=20 addition it was subject to a lawsuit that it had not received sufficient = review. The suit failed = and the=20 first authorized test of humkan gene gransfer began on 22 May 1989.

The p53 protein is a tumor suppressor that has = sequence=20 specific DNA binding activity and contains a domain that can activate=20 transcription when attached to the DNA binding domain of another = protein. The encoding gene for p53 has = been found=20 to be mutated in a variety of human cancers. The normal p53 activates the = expression=20 of a gene that has a p53 binding site and that activation correlates = with the=20 ability of p53 to bind DNA. = Cancer=20 causing forms of p53 do not activate transcription and inhibit = activation by=20 normal p53. Thus = oncogenic forms of=20 p53 may promote tumor formation by interfering with normal p53 mediated=20 activation of genes involved in growth inhibition.

In May 1993 the sequence of chromosome 3 of the = yeast=20 Sacramyces cervesa, consisting of 315,357 base pairs was published in = the=20 magazine Nature in a paper with 147 co authors. This is the largest continuous = stretch=20 of DNA ever sequenced. It = is packed=20 with genes whose functions are completely unknown. The sequencing was funded by = the=20 EC. At about about 35 = different=20 labs. The total yeast genome of 16 chromosomes and 14 million base pairs = is=20 almost totally devoid of pseudogenes and repetitive sequences. The chromosome 3 sequence = contains 182=20 open reading frames coding for proteins of over 100 amino acids in = length. It is=20 surprising that a large fraction fail to show significant homology to = the 35,000=20 or so genes deposited in various data bases. Determining the function of = these genes=20 is difficult, time consuming work. =20 For example, Piotr Slonimski in France found a 6.5 kb open = reading frame=20 had no effect on the yeast under normal conditions. But the gene turned out to be = essential=20 to resist killing by acid and low pH. =20 At pH 4.5 there was an effect, and at 4.0 absence of the gene was = lethal. With genes of = unknown=20 function it is difficult to decide what conditions to look for such as=20 morphology, temperature, sporulation, etc.

Among the genes showing homology to known genes is = one=20 similar to part of the nitrogen fixation operon of bacteria. Although the yeast does not = fix=20 nitrogen, the gene is essential for growth.

Another problem that can be explored using the = yeast=20 chromosome 3 genetic information is why there are recombination "hot=20 spots". Comparison of = sequence data=20 with genetic map data could help determine factors that help determine=20 recombination frequency.

The first generation of biotechnology products were = predominately genetically engineered proteins that worked more or less = like=20 their natural counterparts such as recombinant variations of human = insulin and=20 growth hormone.

Plant biotechnology. =20 Researchers around the world have genetically engineered plants = to=20 manufacture a wide variety of materials including human proteins such as = albumin=20 and interferon. They have = even=20 induced plants to manufacture a type of polyester. Much plant work depends on = gene transfer=20 technology developed for plants at the Max Plank institute for plant = breeding in=20 Cologne Germany, the state university of Ghent, Belgium, the state = university of=20 Leiden in the Netherlands and Monsanto Corporation in St. Louis. A modified form of the Ti = plasmid, a=20 piece of DNA from the bacteria Agribacteria tumefaciens that naturally = infects=20 plant cells is an efficient vector for carrying new genes into plants. = Ti=20 plasmids have been used to create many strains of transgenic plants.

The first area of application for genetically = engineered=20 plants was improved traits such as disease resistance. The second area was = improved=20 processing traits such as ripening in tomatos. Now plants are being used to = produce=20 specialty chemicals.

Cystic Fibrosis. =20 CF is the most common potentially lethal autosomal disease of = Caucasians=20 affecting one in 2500 newborns. = It=20 is a recessive gene meaning that persons with one normal and one mutant = allele=20 are asymptomatic. This = suggests=20 that the population is heterozygous in 1 in 50 individuals. However, some studies have = shown that as=20 many as 1/25 individuals carry the gene. =20 This means that the error may be combined with other errors and = yield=20 spontaneous abortions or lack of fertilization 3/4th of the time. Infertility is almost = universal in CF=20 males and frequent in females. =20 Survival has improved tremendously with the current median = survival being=20 about 30 years and an individual born today with CF would be expected to = survive=20 about 40 years with current therapy. =20 CF was named by Anderson in 1938. =20 In the early 1980's it was demonstrated that the normal efflux of = chloride ions across respiratory epithelial cell membrane in response to = cyclic=20 AMP is lacking in patients with CF. =20 However this information was not sufficient to identify the = protein=20 product of the gene that is defective in CF patients.

In 1989 the CF gene was identified. Initially it was mapped to = chromosome 7=20 using linkage analysis of individuals and polymorphic DNA markers. Then subsequent genetic = analysis placed=20 the gene in a 1.5 megabase range on chromosome 7. Using chromosome jumping and = chromosome=20 walking, the gene itself was identified. =20

The CF gene is about 250,000 bp long and it's = messenger RNA=20 transcript is about 6,500 bases which encodes a protein of 1,480 amino=20 acids. A deletion in this = gene=20 resulting in the loss a single amino acid (phenylalanine at codon 508) = was=20 identified in CF patients. = This=20 delta f 508 accounts for approximately 70% of CF cases. The protein was named CFTR the = Cystic=20 Fibrosis Transmembrane Conductance Regulator. Over 170 other mutations in = the CFTR=20 gene have been described for individuals with CF. About 20 of these are commonly = occurring=20 among caucasions. The = ability to=20 detect the major CF mutations makes it possible to detect about 90% of = the CF=20 carriers. This raised the = possiblity of population screening for CF to identify couples at risk = and=20 provide them with genetic counseling.

Walter Gilbert of Harvard University proclaimed = that=20 "sequencing the human genome is like pursuing the Holy Grail."

In 1984 the Alta meeting sponsored by DOE at Alta = Utah,=20 mention was made of sequencing the whole human genome. In 1985 Robert Sinshimer, = chancellor of=20 UC Santa Cruz held a meeting to discuss such a project. In March 1986 Delbecco of the = Salk=20 Institute wrote that sequencing the human genome would be a turning = point in=20 cancer research. Also in = March 86=20 Charles DeLisi proposed that DOE laboratories should be the center of = the genome=20 effort after the Santa Fe conference in March 1986. In 1983 DOE established the = GeneBank at=20 the Lawrence Livermore Laboratory.

In 1986 the Cold Spring Harbor Symposium on = Molecular Biology=20 of Homo Sapiens, James Watson expressed concern over the prospect of DOE = being=20 so deeply involved in a biological project and proposed that the project = be=20 centered at NIH.

In August 1986 the National Research Council began = 14 months=20 of deliberation which ended in their urging the US congress to begin the = human=20 genome project and invite other nations to pursue this as a common = effort. The OTA prepared a report in = 1988. It noted that "there is no = single human=20 genome project, but instead many projects". In 1987 Leroy Hood proclaimed = "the=20 sequence of the human genome would be perhaps the most powerful tool = ever=20 developed to explore the mysteries of human development and = disease". I would suggest it is a = resource not a=20 tool. In 1990 James = Watson wrote in=20 Science Magazine "The United States has now set as a national objective = the=20 mapping and sequencing of the human genome".

The goals for 1990 to 1995 are:

Genetic linkage maps

Physical maps

Improved DNA sequencing technology

Although we are separated by at least 30 million = years of=20 evolution our chromosomes differ from those of the chimpanzee in only = about 1%=20 of the genes.

Big science projects Manhattan project, apollo, =

Interesting legal ramifications arise in the = patents of=20 discoveries and techniques. = The=20 Gallo case, the methodology for detecting the CF gene, Ventner's NIH = patent=20 application.

An interesting analogy, aside from the stadium as = nucleus=20 analogy would be the amount of material in the genes of a cell compared = to the=20 amount of material in the cell. =20 Compare it with the amount of dirt removed to get a single = diamond.

Each of the 23 human chromosome pairs assumes a = unique shape=20 during cell division (mitosis). =20 This permits one to prepare a karyotype of human chromosomes=20 photographing the chromosomes from a dividing cell and arranging them in = 23=20 separate pairs. Human = chromosomes=20 are about 60% protein and 40% DNA. =20 About every 200 nucleotides the chromosomal DNA helix is wrapped around a = complex of=20 small polypeptides called histones. =20 These proteins are positively charged and attracted to the = negatively=20 charged DNA.

Between 10 and 25% of human DNA is short sequences = of tandem=20 repeats. These tandem = repeats are=20 located at the centromeres where the two chromosomes remain attached = after=20 replication.

Humans have extensive intervening sequences called=20 introns. Introns are = removed from=20 messenger RNA in a process called RNA splicing. Bacteria do not have = introns.

Normally only about 1% of the human genome is = expressed in=20 any cell. Promoter = sequences in=20 bacteria and eukaryotes are different. =20 To achieve expression of eukaryotic DNA in bacteria, a bacterial=20 ribosomal binding site must be added.

The four basic steps in cloning human genes = are:

1. make fragments

2. join fragments to vector

3. introduce recombinant into host cell

4. screen or select cells that have acquired the=20 recombinant.

Koshland's article in science for the Human Genome = 91 meeting=20 says "There is the immorality of omission, the failure to apply the = technology=20 to aid the poor, the infirm, and the underprivileged" as well as "the = immorality=20 of commission"

Big Science Little Science - How can good = scientific research=20 be accomplished in big chunks? = How=20 should this work be funded. = Watson=20 said "We want it big enough so the costs can be brought down to a = reasonable=20 level" and "we want it done in a reasonable period of time"

Three billion is not necessarily that much. The SSC in Texas will cost 8=20 billion. The planned = Space Station=20 will cost 40 billion to construct and 80 billion to operate. What was = the cost=20 of the Manhattan project, B1 bomber R&D

Actually much of the human genome project is = technology not=20 research. It is = drudgerous=20 application work

Setting up a small mol biol lab costs over = 100,000

Each trident sub costs 1.5 billion

The stealth bomber program cost 68 billion

SDI funding for FY 88-92 is 38 billion

Over 6 billion per year is spent for US nuclear = warheads

If human genome is 3 billion base pairs yet only = 50,000=20 proteins.

The average protein requires only a couple thousand = base=20 pairs to code for it. So = 80 - 95%=20 of the base pairs do not code for proteins. At the first Human Genome = Meeting some=20 argued that analyzing this "junk DNA" was a waste of effort. "Let's go for the protein = coding genes"=20 they said. More and more = importance=20 has been attached to this additional non-coding DNA. In fact the percentage of = "junk DNA"=20 seems to fall each year.

HUGO - history, alta, gilbert, dulbecco, big = science,=20 players, 5 year plans

Junk DNA

Genes CF Huntington=92s disease, alcoholism, = perfect pitch,=20 cancer

Model organisms

e. coli, yeast, worm, plants, mouse other

Structure, Program, Status

WHAT IS THE HUMAN GENOME PROJECT

Junk DNA

The genome, rather than being a catalog of 100,000 = or so=20 genes interspersed with junk, is now viewed as a complex "information = organelle"=20 full of control and maintenance subsytems.

In prokaryotes the genome is almost entirely made = up of genes=20 that code for particular proteins, with some transfer and messenger RNA = genes=20 added.

In the center and at the ends (telemeres) of = chromosomes is=20 long stretches of satellite DNA which appears to bind proteins that stop = the=20 ends of chromosomes from fraying or breaking apart.

Most evolutionary theorists believe that introns = are the=20 remnants of primitive RNA life forms that became incorporated into cells = and=20 evolved with them.

A current catalog of "junk" DNA would include:

Introns - sequences within protein coding genes = that do not=20 code for protein.

Small nucleolar RNAs are encoded by some introns = and may play=20 a part in ribosome assembly.

Satellites are short DNA sequences repeated = hundreds or=20 thousands of times at the center and ends of chromosomes. They may play a role in = maintaining=20 chromosomal structural integrity.

Minisatellites and microsatellites are repetitive = sequences=20 that are found throughout the genome. =20 Defects in these repetitive sequences may be associated with = diseases and=20 cancer.

3' untranslated regions occur after the final = protein coding=20 regions of genes and may contain sequences that regulate gene = activity.

Heterogeneous nuclear RNAs are sequences translated = from DNA=20 some of which code for genes but the function of most is unknown.

Long interspersed elements up to 7,000 base = pairs.

Pseudogenes are sequences that would code for = proteins but do=20 not include introns and are not usually expressed in eucaryotes.

In hindsight the first call to sequence the human = genome was=20 from Victor McKusik (Mendelian Inheritance in Man).

Walter Gilbert at Harvard proclaimed that = "sequencing the=20 human genome is like pursuing the holy grail".

Jim Watson suggested that the project be centered = at=20 NIH. The NAS urged = Congress to=20 begin the HGP. In 1988 = the OTA=20 prepared a report informing Congress of the options.

Genetic linkage maps. =20 Physical maps. = McKusicks=20 book.

By 1990 sequencers handled 10,000 bases per day at = a cost of=20 about $5 per base sequenced. = Today=20 that figure has more than doubled and the cost is below $1 per base.

June 26, 2000 President Clinton announced =

PRESIDENT CLINTON ANNOUNCES THE COMPLETION OF THE FIRST SURVEY =
OF THE ENTIRE HUMAN GENOME

   Hails Public and Private Efforts Leading to =
This Historic Achievement

           &n=
bsp;           &nb=
sp;       June 26, =
2000

Today, at a =
historic White House event with British Prime Minister Tony Blair, =
President Clinton announced that the international Human Genome Project =
and Celera Genomics Corporation have both completed an initial =
sequencing of the human genome -- the genetic blueprint for human =
beings.  He congratulated =
the scientists working in both the public and private sectors on this =
landmark achievement, which promises to lead to a new era

of =
molecular medicine, an era that will bring new ways to prevent, =
diagnose, treat and cure disease.  The President pledged to continue and accelerate the =
United States' commitment to helping translate this blueprint into novel =
healthcare strategies and therapies.  He will underscore that this genetic information must =
never be used to stigmatize or discriminate against any individual or =
group.  Our scientific =
advances must always incorporate our most cherished values, and the =
privacy of this new information must be protected.

President=20 Clinton: =93Nearly two centuries ago, in this room, on this floor, = Thomas=20 Jefferson and a trusted aide spread out a magnificent map -- a map = Jefferson had=20 long prayed he would get to see in his lifetime. The aide was Meriwether = Lewis=20 and the map was the product of his courageous expedition across the = American=20 frontier, all the way to the Pacific. It was a map that defined the = contours and=20 forever expanded the frontiers of our continent and our imagination.

=93Today,=20 the world is joining us here in the East Room to behold a map of even = greater=20 significance. We are here to celebrate the completion of the first = survey of the=20 entire human genome. Without a doubt, this is the most important, most = wondrous=20 map ever produced by humankind.=94

Bill=20 Hazeltine remarked that like the man on the moon achievement of NASA = signified=20 our interest in space, this signifies our interest in genomics

Craig=20 Ventner

In=20 youth was a serious surfer. = Medical=20 corpsman in Da Nang Viet Nam sobered by suffering and became = doctor. Switched to research on genes = that code=20 for adrenalin reseptors in heart muscles.

Leroy=20 Hood at Cal Tech and Michael Hunkapillar deeoped sequencing = machine. Hunkapillar of Applied = Biosystems built=20 sequence4s. Used = florescent dyes=20 rather than radioactive tracers.

At=20 NIH Ventner=92s lab got Applied Biosystems sequencers.

Studied=20 EST (Expressed Sequence Tags) the expressed messenger RNA and found = hundreds of=20 previously unknonwn genes in human brain cells.

Bernadine=20 Healy director of NIH decided to apply for patents. Ventner told congress that NIH = was=20 submitting patents on brain genes.

Watson=20 was against patenting potential gene sequences. Patent issue created problem = with Healey=20 who took petty bureaucratic actions against Watson, raised baseless = financial=20 conflict of interest questions etc and led to Watson=92s resignation in = April=20 1992.

Ventner=20 was pawn of Healy (he had no stake in patent issue). NIH rejected Ventners requests = for funds=20 to sequence part of human genome.

Wallace=20 Sternberg, a venture capitalist and chairman of Health Care Investment = set up=20 Human Genome Science and provided $70 million to TIGR (The Institute for = Genomic=20 Research) to be run by Ventner.

William=20 Hazeltine was president of Human Genome Science. In July 1992 Ventner left NIH = taking=20 most of his staff.

Ventner=20 captured EST=92s for mouse and human genes. =20 HGS required that anyone publishing based on these sequences had = to show=20 the manuscript to HGS in case it wished to patent anything.

In=20 1993 Hamilton Smith ) Nobel Prize 1978 for Restrictive Enzymes) = suggested to=20 Ventner that they sequence the genome of a bacteria. (in 1992 Sulston and Waterson = published=20 a 100,000 base pair stretch of C elegans genome) E coli is 4.6 million base = pairs. so=20 Smith proposed Haemophilus influenza (no relation to the disease) genome = of 1.8=20 million bases. Smith = proposed using=20 the shotgun method.

In=20 May 1995 Ventner announced that he had sequenced the entire H influenza=20 genome. It is interesting = that he=20 had 90% done before NIH rejected his grant because the approach = (shotgun) was=20 not likely to succeed and not worth supporting.

In=20 1995 Sternberg died Ventner and Hazeltine grew further apart. Ventner was interested in = comparative=20 genomics. Hazeltine was = after EST=20 products that might yield drugs. = In=20 1997 they divorced HGS = and=20 TIGR. Ventner renounced = $38 million=20 obligation of HGS to TIGR.

Manhattan=20 project

Apollo=20 project

Weapons=20 projects Billions for = atomic=20 submarines, and even more than the cost of construction for = disposal.

In=20 1970 if you wanted to understand the gene for hemoglobin, you might want = 50=20 milligram (1/1,000th of an ounce) for analysis. But since the gene is only one = of a=20 million or so, this would represent perhaps 1,500 base pairs in the=20 3,000,000,000 base pairs of the human genome. If you started with raw human = DNA and=20 could separate out the gene without loss you would need 3,000,000,000 / = 1,500 =3D=20 2,000,000 which divided by 1,000 still leaves two thousand ounces or = over 100=20 pounds of human DNA. This = would=20 entail perhaps 50,000 pounds of human cells.

The=20 Human genome project is not really a big science project in the sense = that the=20 Manhattan Project or the Superconducting Super Collider or the Hubble = telescope=20 are big science projects for several reasons.

First=20 the project is not that big. = The=20 total proposed effort is on the order of 3 billion dollars over 15 = years.

Second=20 the project is not to be performed by one organization. There will be many groups = contributing=20 to the effort.

Third=20 the project is really a collection of different projects. These projects are:

1.=20 Establishment and maintenance of databases containing information about = nucleic=20 acid and protein sequences from various different organisms.

2.=20 Creation of maps of human chromasomes. =20 Maps are DNA markers that would permit scientists to derive the = rough=20 location of a particular gene rapidly.

3.=20 Create repositories of research materials such as sets of DNA fragments = which=20 fully represent the DNA in the human chromosomes.

4.=20 Development of instruments and technology to analyze DNA, proteins, = structures,=20 etc.

5.=20 Non-human DNA libraries and resources, and

6.=20 Actual sequencing of all or part of the human genome and that of other=20 organisms.

WHO=20 ARE THE PLAYERS?

NIH=20 spends about 6 billion per year on biomedical research. Of this about 20 million is = for genome=20 work.

DOE=20 spends 230 million in biological research of which about 15 million is = for=20 genome work. DOE = rationalizes their=20 entry into the field due to work on biological effects of = radiation. DOE has worked on development = of much of=20 the new equipment for genome work.

NSF=20 is currently a negligible player but is a logical organization to = support=20 technology development.

HHMI=20 is a private organization spending 20 million per year on genetics and 2 = million=20 on data base work.

Welcome=20 Trust of London

Watson forged genome consortium, US = Britian=20 France Germanyy China Japan = daily=20 publication of results, public domain spread money to several = institutions=20 believed NIH should do it genomes of other organisms E Coli C elegans=20 Drosophilia, Mouse, etc.

Foreign=20 countries have devoted rather small amounts to human genome work = although work=20 is being done in France, England, and Japan.

The=20 genome project will require significant innovation in the ability to = manipulate=20 DNA. Major advances in = analytic=20 instrumentation would be required for cost effective ordering and = sequencing of=20 segments. Theoretical = developments=20 in computer science and mathematical biology and great expansion in the = ability=20 to store and manipulate the information and interface it with other = large and=20 diverse genetic databases would be required.

THE=20 HUMAN GENOME

The=20 human genome is made of forty-six chromosomes, long strands of DNA. Each strand of DNA is made of = subunits=20 called nucleotides. There = are three=20 billion pair of nucleotides in the human genome. Twenty three chromosomes are = inherited=20 from each parent. The = amount of=20 information carried by these chromosomes is immense. Each nucleotide position can = be any of=20 four different nucleotides. = In=20 computer terms this would be two bits of information. Today's best computer chips = hold ten=20 million bits of information on a chip one millimeter by one millimeter = by .00001=20 millimeters. This is = twenty=20 thousand times the volume of the nucleus of a human cell so the computer = chip=20 stores five hundred bits of information in the same space as the nucleus = stores=20 the human genome. The = human genome,=20 by contrast, stores six billion bits of information or more than ten = million=20 times as much information in the same area.

Three=20 nucleotides code for a single amino acid. =20 The average protein might be about 500 amino acids long. This would mean that the human = genome=20 could code for two million different proteins. In actuality the genome codes = for only=20 about 60,000.

Genes=20 make up an organism's genotype. = Its=20 appearance, function and behavior make up its phenotype. Genes and the environment work = together=20 to shape the phenotype.

There=20 is no such thing as the single definitive "human genome". Every gene in the human gene = pool comes=20 in multiple forms perhaps differing in only a single nucleotide, called=20 alleles. Some alleles are = for=20 rather different characteristics such as the blood type genes. Some individuals have the = 'type A'=20 allele, others the 'type B', others are heterozygous with blood type = 'AB' and=20 some 'type O' have neither. = The=20 gene pool of the cheetah is extremely small. This has been shown by Steve = O'Brian at=20 NIH who found that skin grafts between unrelated cheetahs lasted for = many weeks=20 and often were not rejected at all. =20 This is in contrast to grafts between other cats (or other = species) which=20 are normally rejected in a matter of days to a couple weeks. I am impressed with the work = done on=20 saving the Condor. = Researchers in=20 San Diego evaluated the genome of available condors to select mates with = genetic=20 diversity in breeding condors. = Many=20 killi fish fanciers are concerned about the possible loss of species due = to the=20 destruction of the rain forests and other killifish habitats. We have begun to explore how = many=20 individuals must be kept to maintain a reasonable genetic diversity.

While=20 thinking about cheetahs, the breeding program at the Wildlife Safari in = Oregon=20 was devastated by an infection of feline peritonitis (which is common = among=20 house cats). Almost half = of the=20 cheetahs died from the disease whereas nearby lions were barely = affected. It appears that cheetahs in = the wild are=20 solitary and unlikely to encounter the disease. Resistance was not present in = the=20 limited cheetah genome. = However=20 cheetahs in zoos are likely to encounter the disease and it can be=20 devastating. The behavior = of=20 humans, in transferring and introducing organisms from place to place = and=20 increasing communication are likely to increase the danger of harmful = diseases=20 being introduced and spread beyond their natural containment. Another example is = malaria. The benign tertian malaria is = almost=20 nonexistent in equatorial Africa because almost all natives are=20 "Duffy-negative". The = Duffy antigen=20 is a protein-carbohydrate complex on red blood cells that is necessary = for the=20 malaria parasite to attack humans. =20 In fact malaria was practically driven out of the tropics by the=20 resistance of the population. =

Structure=20 function

SHOULD=20 $3 BILLION BE DEVOTED TO THE HUMAN GENOME PROJECT?

One=20 critic has said that sequencing the complete human genome is like = translating=20 Shakespear into Sanscrit.

This=20 is based on the fact that knowing the sequence of the 3 billion = nucleotides that=20 make up the human genome doesn't tell us anything about the proteins = they code=20 for, the mechanisms of enhancement and repression, etc.

One=20 of the biggest problems facing molecular biologists is the problem of=20 determining the structure of a protein from a description of its = sequence.

Daniel=20 Koshland in a Science editorial says failure to sequence the human = genome is=20 "the equivalent of providing iron lungs to polio victims at the expense = of=20 working on a vaccine".

Commercial=20 implications include availability of data. =20 Should human genome sequences be subject to copyright?

While=20 ethical and social questions presented by human genome mapping are = similar to=20 those addressed in other settings such as genetic counseling, the scale = "the=20 complete biological book on humankind" is much greater. Its like saying we've always = had the=20 problem of weapons, so what's different about the atom bomb?

As=20 mapping work proceeds large family histories will be studied. Confidentiality problems = exist. Should confidentiality be = broken when=20 the probability is high that serious, avoidable harm would otherwise = come to=20 identifiable individuals?

What=20 about people with HLA-B-27? = This is=20 a human leukocyte antigen that is associated with the disease ankylosing = spondylitis. However only = one in a=20 thousand people with the marker get the disease.

Currently=20 genetic counseling and screening is limited to such factors as Down's = syndrome=20 and a few major genetic diseases. =20 Are there limits on the traits that parents should be able to = decide=20 there children must have?

MOUSE=20 MODELS FOR AIDS

In=20 1942 Avery combined two viruses in the same cell. Some of them exchanged = coats. This is known as = pseudotyping.

Mice=20 have been developed with a severe combined immunodeficiency (SCID). These mice have a genetic = defect that=20 has wiped out the mouse's own immune system. These animals are then grafted = with a=20 human immune system. SCID = mice are=20 used in AIDS studies. = They are=20 infected with HIV-1 (Human Immunodeficiency Virus 1). This permits researchers to = determine=20 what happens with the AIDS virus in human cells in living animals. This is more useful than = studies in=20 cultured cells only. An = example of=20 the use of these models is the finding that AIDS virus infected mice = respond to=20 the antiviral drug AZT. = This means=20 that the mouse model may be of use in AIDS drug evaluation.

Robert=20 Gallo at NIH has reported that the AIDS virus can react with a common = mouse=20 virus when they are both in the same cell. =20 It appears that AIDS viruses coated with Murine Leukemia Virus = coats have=20 been produced. These new = viruses=20 have some disturbing characteristics. =20 First they reproduce much faster than the AIDS virus. Second they are able to infect = types of=20 cells that are immune to the AIDS virus.

The=20 possibility exists of producing viruses that can spread by different = routes such=20 as being transmitted throught the air.

HUMAN=20 GENE TRANSFER TEST

In=20 1988 Dr. Stephen Rosenberg of the National Cancer Institute began an = experiment=20 involving injection of genetically modified tumor-infiltrating = lymphocytes (TIL=20 cells) into human patients.

Rosenberg=20 has had success in treating melanomas with TIL cells that have been = cultured=20 with interleukin-2 a growth factor that stimulates the immune = system. This only works in a fraction = of the=20 patients. Why? The gene transfer experiment = is an=20 attempt to develop a better understanding of what is happening. Neomycin resistance can be = easily traced=20 since it is not normal to human cells.

In=20 the gene transfer experiment, the patient's own lymphocytes were = extracted and=20 labeled with a bacterial "marker" gene (resistance to the antibiotic=20 neomycin). A retrovirus = containing=20 the marker was used as the delivery system. Then the cells were cultured = to produce=20 a large quantity of cells which were reintroduced into the patient's=20 bloodstream.

Results=20 reported to date are: that the gene labeled TIL cells did go to the = tumor; that=20 the cells survived in the body; and that adding the marker did not = change their=20 characteristics.

If=20 data on the initial five patients continue to suggest a connection = between TIL=20 cells homing in on tumors and tumor shrinkage, the next step is to = identify=20 which of the multiple types of cells in the TIL cell preparation are = doing the=20 job.

In=20 the future it might be possible to use TIL cells as a mechanism to get=20 anticancer agents into a tumor. = The=20 neomycin resistance marker doesn=92t have any therapeutic effect but it = might be=20 possible to use cells with an anticancer agent. For example the gene for human = tumor=20 necrosis factor has been transferred to human cells in culture.

The=20 gene transfer experiment was reviewed a total of 15 times before = receiving final=20 approval. In addition it = was=20 subject to a lawsuit that it had not received sufficient review. The suit failed and the first = authorized=20 test of human gene transfer began on 22 May 1989.

The=20 p53 protein is a tumor suppressor that has sequence specific DNA binding = activity and contains a domain that can activate transcription when = attached to=20 the DNA binding domain of another protein. =20 The encoding gene for p53 has been found to be mutated in a = variety of=20 human cancers. The normal = p53=20 activates the expression of a gene that has a p53 binding site and that=20 activation correlates with the ability of p53 to bind DNA. Cancer causing forms of p53 do = not=20 activate transcription and inhibit activation by normal p53. Thus oncogenic forms of p53 = may promote=20 tumor formation by interfering with normal p53 mediated activation of = genes=20 involved in growth inhibition.

In=20 May 1993 the sequence of chromosome 3 of the yeast Sacramyces cervesa,=20 consisting of 315,357 base pairs was published in the magazine Nature in = a paper=20 with 147 co authors. This = is the=20 largest continuous stretch of DNA ever sequenced. It is packed with genes whose = functions=20 are completely unknown. = The=20 sequencing was funded by the EC at about 35 different labs. The total = yeast=20 genome of 16 chromosomes and 14 million base pairs is almost totally = devoid of=20 pseudogenes and repetitive sequences. =20 The chromosome 3 sequence contains 182 open reading frames coding = for=20 proteins of over 100 amino acids in length. It is surprising that a = large=20 fraction fail to show significant homology to the 35,000 or so genes = deposited=20 in various data bases. = Determining=20 the function of these genes is difficult, time consuming work. For example, Piotr Slonimski = in France=20 found a 6.5 kb open reading frame had no effect on the yeast under = normal=20 conditions. But the gene = turned out=20 to be essential to resist killing by acid and low pH. At pH 4.5 there was an effect, = and at=20 4.0 absence of the gene was lethal. =20 With genes of unknown function it is difficult to decide what = conditions=20 to look for such as morphology, temperature, sporulation, etc.

Among=20 the genes showing homology to known genes is one similar to part of the = nitrogen=20 fixation operon of bacteria. =20 Although the yeast does not fix nitrogen, the gene is essential = for=20 growth.

Another=20 problem that can be explored using the yeast chromosome 3 genetic = information is=20 why there are recombination "hot spots". =20 Comparison of sequence data with genetic map data could help = determine=20 factors that help determine recombination frequency.

Cystic=20 Fibrosis. CF is the most = common=20 potentially lethal autosomal disease of Caucasians affecting one in 2500 = newborns. It is a = recessive gene=20 meaning that persons with one normal and one mutant allele are=20 asymptomatic. This = suggests that=20 the population is heterozygous in 1 in 50 individuals. However, some studies have = shown that as=20 many as 1/25 individuals carry the gene. =20 This means that the error may be combined with other errors and = yield=20 spontaneous abortions or lack of fertilization 3/4th of the time. Infertility is almost = universal in CF=20 males and frequent in females. =20 Survival has improved tremendously with the current median = survival being=20 about 30 years and an individual born today with CF would be expected to = survive=20 about 40 years with current therapy. =20 CF was named by Anderson in 1938. =20 In the early 1980's it was demonstrated that the normal efflux of = chloride ions across respiratory epithelial cell membrane in response to = cyclic=20 AMP is lacking in patients with CF. =20 However this information was not sufficient to identify the = protein=20 product of the gene that is defective in CF patients.

In=20 1989 the CF gene was identified. = Initially it was mapped to chromosome 7 using linkage analysis of = individuals and polymorphic DNA markers. =20 Then subsequent genetic analysis placed the gene in a 1.5 = megabase range=20 on chromosome 7. Using = chromosome=20 jumping and chromosome walking, the gene itself was identified.

The=20 CF gene is about 250,000 bp long and it's messenger RNA transcript is = about=20 6,500 bases which encodes a protein of 1,480 amino acids. A deletion in this gene = resulting in the=20 loss a single amino acid (phenylalanine at codon 508) was identified in = CF=20 patients. This delta f = 508 accounts=20 for approximately 70% of CF cases. =20 The protein was named CFTR the Cystic Fibrosis Transmembrane = Conductance=20 Regulator. Over 170 other = mutations=20 in the CFTR gene have been described for individuals with CF. About 20 of these are commonly = occurring=20 among Caucasians. The = ability to=20 detect the major CF mutations makes it possible to detect about 90% of = the CF=20 carriers. This raised the = possibility of population screening for CF to identify couples at risk = and=20 provide them with genetic counseling.

Walter=20 Gilbert of Harvard University proclaimed that "sequencing the human = genome is=20 like pursuing the Holy Grail."

In=20 1984 the Alta meeting sponsored by DOE at Alta Utah, mention was made of = sequencing the whole human genome. =20 In 1985 Robert Sinshimer, chancellor of UC Santa Cruz held a = meeting to=20 discuss such a project. = In March=20 1986 Delbecco of the Salk Institute wrote that sequencing the human = genome would=20 be a turning point in cancer research. =20 Also in March 86 Charles DeLisi proposed that DOE laboratories = should be=20 the center of the genome effort after the Santa Fe conference in March=20 1986. In 1983 DOE = established the=20 GeneBank at the Lawrence Livermore Laboratory.

In=20 1986 the Cold Spring Harbor Symposium on Molecular Biology of Homo = Sapiens,=20 James Watson expressed concern over the prospect of DOE being so deeply = involved=20 in a biological project and proposed that the project be centered at = NIH.

In=20 August 1986 the National Research Council began 14 months of = deliberation which=20 ended in their urging the US congress to begin the human genome project = and=20 invite other nations to pursue this as a common effort. The OTA prepared a report in = 1988. It noted that "there is no = single human=20 genome project, but instead many projects". In 1987 Leroy Hood proclaimed = "the=20 sequence of the human genome would be perhaps the most powerful tool = ever=20 developed to explore the mysteries of human development and = disease". I would suggest it is a = resource not a=20 tool. In 1990 James = Watson wrote in=20 Science Magazine "The United States has now set as a national objective = the=20 mapping and sequencing of the human genome".

The=20 goals for 1990 to 1995 are:

Genetic=20 linkage maps

Physical=20 maps

Improved=20 DNA sequencing technology

Although=20 we are separated by at least 30 million years of evolution our = chromosomes=20 differ from those of the chimpanzee in only about 1% of the genes.

Big=20 science projects Manhattan project, apollo,

Interesting=20 legal ramifications arise in the patents of discoveries and = techniques. The Gallo case, the = methodology for=20 detecting the CF gene, Ventner's NIH patent application.

An=20 interesting analogy, aside from the stadium as nucleus analogy would be = the=20 amount of material in the genes of a cell compared to the amount of = material in=20 the cell. Compare it with = the=20 amount of dirt removed to get a single diamond.

Each=20 of the 23 human chromosome pairs assumes a unique shape during cell = division=20 (mitosis). This permits = one to=20 prepare a karyotype of human chromosomes photographing the chromosomes = from a=20 dividing cell and arranging them in 23 separate pairs. Human chromosomes are about = 60% protein=20 and 40% DNA. About every = 200=20 nucleotides the chromosomal DNA =20 helix is wrapped around a complex of small polypeptides called=20 histones. These proteins = are=20 positively charged and attracted to the negatively charged DNA.

Between=20 10 and 25% of human DNA is short sequences of tandem repeats. These tandem repeats are = located at the=20 centromeres where the two chromosomes remain attached after = replication.

Humans=20 have extensive intervening sequences called introns. Introns are removed from = messenger RNA=20 in a process called RNA splicing. =20 Bacteria do not have introns.

Normally=20 only about 1% of the human genome is expressed in any cell. Promoter sequences in bacteria = and=20 eukaryotes are different. = To=20 achieve expression of eukaryotic DNA in bacteria, a bacterial ribosomal = binding=20 site must be added.

The=20 four basic steps in cloning human genes are:

1.=20 make fragments

2.=20 join fragments to vector

3.=20 introduce recombinant into host cell

4.=20 screen or select cells that have acquired the recombinant.

Koshland's=20 article in science for the Human Genome 91 meeting says "There is the = immorality=20 of omission, the failure to apply the technology to aid the poor, the = infirm,=20 and the underprivileged" as well as "the immorality of commission"

Big=20 Science Little Science - How can good scientific research be = accomplished in big=20 chunks? How should this = work be=20 funded. Watson said "We = want it big=20 enough so the costs can be brought down to a reasonable level" and "we = want it=20 done in a reasonable period of time"

Three=20 billion is not necessarily that much. =20 The SSC in Texas will cost 8 billion. The planned Space Station will = cost 40=20 billion to construct and 80 billion to operate. What was the cost of the = Manhattan project, B1 bomber R&D

Actually=20 much of the human genome project is technology not research. It is drudgerous application = work

Setting=20 up a small mol biol lab costs over 100,000

Each=20 trident sub costs 1.5 billion

The=20 stealth bomber program cost 68 billion

SDI=20 funding for FY 88-92 is 38 billion

Over=20 6 billion per year is spent for US nuclear warheads

If=20 human genome is 3 billion base pairs yet only 50,000 proteins.

The=20 average protein requires only a couple thousand base pairs to code for = it. So 80 - 95% of the base pairs = do not=20 code for proteins. At the = first=20 Human Genome Meeting some argued that analyzing this "junk

DNA"=20 was a waste of effort. = "Let's go=20 for the protein coding genes" they said. =20 More and more importance has been attached to this additional = non-coding=20 DNA. In fact the = percentage of=20 "junk DNA" seems to fall each year.