Prof. Dr. ANIL KUMARSCHOOL OF BIOTECHNOLOGY

DEVI AHILYA UNIVERSITYKHANDWA ROAD

INDORE-452001, INDIAEmail: ak_sbt@yahoo.com

HUMAN GENOME

INTRODUCTION

• The human genome is the genome of Homo sapiens, which is composed of 23 distinct pairs of chromosomes (22 autosomal+X+Y) with a total of approximately 3 billion DNA base pairs containing an estimated 20,000-25,000 genes.

• The Human Genome Project has produced a reference sequence of the euchromatic human genome, which is used worldwide in biomedical sciences.

• The human genome had fewer genes than expected, with only about 1.5% coding for proteins, and the rest comprised by RNA genes, Regulatory sequences, introns and controversially so- called junk DNA.

• In Dec. 1984 during a workshop on current state of mutation detection and characterization and to project future directions for technologies to tackle the prevailing technical limitations, scientists discussed about the human genome analysis and this was the first step towards nucleotide sequencing of the entire human genome. This workshop was being sponsored by the U.S Department of Energy.

• In the workshop, growing roles of existing DNA technologies especially the emerging Gene Cloning and Sequencing technologies were discussed. It was realized that existing technologies are in use for about a decade and mostly individual scientists are engaged in cloning and characterization of single genes which looked to be wasteful of Human and Research resources.

• Such methodologies were considered to be incapable of determining mutations with good sensitivity. Scientists thought that an exhaustive, complex and expansive project for complete nucleotide sequencing of the human genome should be undertaken.

• An idea for a dedicated human genome project by the U.S Department of Energy (DAE) was initiated by the report on technologies for detecting heritable mutations in human beings.

• In 1986, an international meeting in Mexico to assess the desirability and feasibility of ordering and sequencing DNA clones representing the entire human genome was sponsored by DAE.

FEATURES

CHROMOSOMES

• There are 24 distinct human chromosomes with : 22 autosomal chromosomes, plus the sex determining X and Y chromosomes.

• Chromosomes 1-22 are numbered roughly in order of decreasing size.

• Somatic cells usually have one copy of chromosomes 1-22 from each parent , plus an X chromosome from the mother, and either an X or Y chromosome from the father, for a total of 46.

GENES

• There are an estimated 20,000-25,000 human protein coding genes.

• The number of human genes seems to be less than a factor of two greater than that of many such simpler organisms, such as the roundworm and fruit fly.

• Most human genes have multiple exons, and human introns are frequently much longer than the flanking exons.

REGULATORY SEQUENCES

• The human genome has many different regulatory sequences which are crucial to controlling gene expression.

• Identification of regulatory sequences relies in part on evolutionary conservation. The evolutionary branch between the human and mouse, for example, occurred 70-90 million years ago. So computer comparisons of gene sequences that identify conserved non-coding sequences will be an identification of their importance in duties such as gene regulation.

• Another comparative genomic approach to locating regulatory sequences in humans is the gene sequencing of the puffer fish. These vertebrates have essentially the same genes and regulatory gene sequences as humans, but with only one-eighth the “junk” DNA. The compact DNA sequence of the puffer fish makes it much easier to locate the regulatory genes.

OTHER DNA

• Protein coding sequences comprise less than 1.5% of the human genome.

• Aside from genes and known regulatory sequences, the human genome contains vast regions of DNA the function of which is not known.

• These regions comprise the vast majority, by some estimates 97%, of the human genome size.

Much of this is comprised of:-

REPEAT ELEMENTS

• Tandem repeats: Satellite DNA Minisatellite Microsatellite

• Interspersed repeats: SINEs LINEs

TRANSPOSONS

• Retrotransposons 1. LTR (a) Ty1-copia (b) Ty3-gypsy

2. Non- LTR (a) SINEs (b) LINEs

• DNA Transposons

PSEUDOGENES

• There is large amount of sequence that does not fall under any known classification.

• Recent experiments using microarrays have revealed that a substantial fraction of non-genic DNA is in fact transcribed into RNA, which leads to the possibility that the resulting transcripts may have some unknown function.

GENETIC DISORDERS

• The genetic disorders are caused by abnormal expression of one or more genes that matches a clinical phenotype.

• The disorder may be caused by a gene mutation, an abnormal number of chromosomes, or triplet expansion repeat mutations. Defective genes can be inherited from the parents, in which case it is known as a hereditary disease.

• There are around 4,000 known genetic disorders, with the most common being cystic fibrosis.

• Studies of genetic disorders is often performed by means of population genetics.

• Treatment is performed by a geneticist- physician trained in clinical genetics.

• The results of Human Genome Project are likely to provide increased availability of genetic testing for gene related disorders, and eventually improved treatment.

• One major gross effect on human phenotypes derives from gene dosage, whose effects play a role in disorders caused by duplication, omission, or disruption of chromosomes. For example, those afflicted with Down syndrome, or trisomy 21, experience high rates of Alzheimer’s disease, an effect thought to be related to the overexpression of the Alzheimer’s- related amyloid precursor protein whose gene is located on chromosome 21. By contrast, Down’s syndrome sufferers experience lower rates of breast cancer, possibly due to the overexpression of a tumor-suppressor gene.

HUMAN GENOME PROJECT

• The Human Genome Project (HGP) is an international scientific research project.

• The project also focused on several other nonhuman organisms such as Escherichia coli, the fruit fly, and a laboratory mouse.

• It was one of the largest investigational projects in modern science.

• The project began in 1990 initially headed by James D. Watson. A working draft of the genome was released in 2000 and a complete one in 2003, with further analysis still being published.

• The mapping of human genes is an important step in the development of medicines and other aspects of health care.

• The “genome” of any given individual ( except for identical twins and cloned animals ) is unique; mapping “the human genome” involves sequencing multiple variations of each gene.

• The project did not study all of the DNA found in human cells; some heterochromatic areas (about 8% of the total) remain un-sequenced.

• An important feature of HGP was the federal government’s long-standing dedication to the transfer of technology to the private sector.

• By licensing technologies to private companies and awarding grants for innovative research, the project catalyzed the multibillion- dollar U.S. biotechnology industry and fostered the development of new medical applications.

GOALS OF THE HUMAN GENOME PROJECT

Identify all the approximately 20,000-25,000 genes in human DNA.

Determine the sequences of the 3 billion chemical base pairs that make up human DNA.

Store this information in databases.

Improve tools for data analysis.

Transfer related technologies to the private sector.

Address the ethical, legal, and social issues (ELSI) that may arise from the project.

Understand the genetic make up of the human species.

DAE’S THREE MAJOR OBJECTIVES

• Implementation of this program began with a small number of pilot projects.

Generation of refined physical maps of human chromosomes.

Development of support technologies and facilities for human genome research.

Expansion of communication networks and of computational and database capabilities.

OTHER ORGANIZATIONS/COUNTRIES

• In 1988, office of the human genome research was set up with the support from the US office of Technology Assessment and National Research Council and US National Institutes of Health (NIH). It was later renamed as the National Center for Human Genome Research.

• In 1988, US congress approved $3 billion for the project and time limit 15 years commencing from 1991.

• Department Offices of Energy (DOE) funded to number of laboratories like the Lawrence Livermore National Laboratory, the Los Alamos National Laboratory, the Lawrence Berkeley National Laboratory.

• Thereafter, the projects were also started by European countries like Germany, France, Italy, Denmark, The Netherlands, the United Kingdom.

• National Genome Projects were also started by the countries like Australia, Canada, Japan, Korea, New Zealand. That’s way, it became truly an international project.

• Later, Human Genome Organization was established to coordinate the different national efforts, facilitate exchange of research data, public debate etc.

• Three centers of Human Genome Organization were established; HUGO Europe (London); HUGO Americas (Bethesda); and HUGO Pacific (Tokyo).

• Improvement in the DNA sequencing technology became dramatic so that DNA sequencing costs have fallen effectively however without increase in the sequencing efficiency.

HUMAN GENETIC MAPS

• Classic genetic maps for experimental organisms for example, mouse, Drosophila were available since several years and have been refined successively.

• Genetic maps were prepared by crossing different mutants to determine whether the two gene loci are linked or not.

• In human, preparation of genetic map was considered to be based on polymorphic markers which were not necessarily related to disease or to genes. The genetic markers were protein polymorphisms, particularly blood group and serum protein markers.

• In 1980’s, construction of the human genetic maps was considered using restriction fragment length polymorphisms (RFLP’s) solving the problem.

• In 1987, there was first report of human genetic map based on the use of 403 polymorphic loci including 393 RFLP markers.

• Afterwards, high resolution genetic maps have been constructed through the use of microsatellite markers. These markers were more polymorphic than RFLP markers.

• Microsatellites are distributed near the telomeres and not widely distributed throughout the genome. These microsatellites also called as short tandem repeat polymorphisms or SRTPs, are abundant.

• In 1992, second human genetic map was published selecting polymorphic CA/TG repeats, mapping them to specific chromosomes by typing panels of human- rodent somatic cell hybrids and performing statistical linkage analyses on markers from individual chromosomes.

• A total 813 markers were organized into 23 linkage groups.

• Afterwards, maps have been produced with increasing numbers of genetic markers, especially the microsatellite markers and ever increasing resolution. Many different genetic maps have been constructed using different marker sets.

• All the genetic maps have not used the same sets of reference families and relationship between different markers used in genetic maps is not clearly known.

• Nowadays, the most widely distributed set of reference families is that deriving from the Centre d’ Eutdes du Polumorphisme Humaine (CEPH) collaboration of more than 100 independent laboratories. These days integrated genetic maps are prepared.

Advantages of Genetic Maps

• The Genetic maps helped in studying the nature of recombination in humans.

• Besides, it also helped for determination of gene localization.

• The genetic maps also helped in gene cloning.

• The linkage analysis are also helpful in prenatal or presymptomatic diagnosis of inherited disease genes by having many DNA markers in the vicinity of the disease locus.

HUMAN PHYSICAL MAPS

• A physical map will consist of 24 component maps corresponding to 24 chromosomes.

• The first human physical map was obtained using cytogenetic banding technique which distinguished not only the different chromosomes but also discrimination of different sub-chromosomal regions.

• Scientists associated with public Human Genome Project and Celera Genomics published sequences of genome DNA in human.

NATURE ( Feb. 15, 2001) ; SCIENCE (Feb. 16,2001) www.ornl.gov/hgmis/project/journals/journals.html

• Sequence is magnificant and unprecedented resource and is basis for research and discovery throughout this century and beyond.

• Research on Human Genome sequences will have diverse practical applications and impact upon how we feel ourselves and our place in the tapestry of life around us.

• After the entire sequence, the total number of genes in human genome estimated to be nearly 25,000. Some others have estimated upto nearly 30,000-35,000. It is much lower number than the earlier predicted number, nearly 100,000 or even more.

• It is suggested that genetic key to human complexity is not in number of genes but is, how gene parts are translated to synthesize different protein products.

• The earlier concept of one gene one protein is no more. Now, it is estimated that from one gene, even there may be synthesis of upto 20 proteins depending upon the age of the human. It is due to the process of alternative splicing.

• Besides, thousands of post translational chemical modifications are made to proteins, and regulatory mechanisms controlling these processes add to the complexity.

• In constructing the sequence draft, 16 genome sequencing centers produced over 22.1 billion bases of raw sequence data, comprising overlapping fragments totaling 3.9 billion bases. The sequences are sequenced seven times. Over 30% data is high quality, finished sequence with 8-10 fold coverage, 99.99% accuracy and few gaps. All data are freely available via the web (www.ornl.gov/hgmis/project/journals/sequencesites.html)

• Final complete human genome sequence was presented on October 22, 2004.

The following points have been highlighted in the human genome sequence

• Human genome contains 3164.7 million nucleotides.

• Average gene has 3000 nucleotides. Size varies much. Largest known human gene is dystrophin having nearly 2.4 million bases.

• Total number of genes 25000. Much lower number than previously estimated 80,000-140,0000 number. This had been based on extrapolations from gene rich areas as opposed to a composite of gene rich and gene poor areas.

• Almost all ( nearly 99.9%) nucleotide bases are exactly the same in all the persons.

• Functions are unknown for over 50% of the discovered genes.

• Less than 2% of the genome codes for proteins.

• Repeated sequences that do not code for any protein make up at least 50% of the genome. These are called as ‘Junk DNA’.

• Repetitive sequences are thought to have no direct functions but they shed light on chromosome structure and dynamics. It is considered that these repeats reshape the genome by rearranging it creating entirely new genes and modifying and reshuffling existing genes.

• During the last 50 million years, a dramatic decrease seems to have occurred in the rate of accumulation of repeats in the human genome .

• The human genome’s gene dense ‘Urban centers’ are predominantly composed of the DNA building blocks G and C.

• In contrast, the gene poor ‘Deserts’ are enriched in the DNA building blocks A and T. GC and AT rich regions usually can be seen through a microscope as light and dark bands on chromosomes, respectively.

• Genes appear to be concentrated in random areas along the genome with vast expanses of non-coding DNA in between.

• Stretches of upto 30,000 C and G bases repeating over and over often occur adjacent to gene rich areas forming a barrier between the genes and the junk DNA. These CG islands are believed to have role in regulating the gene activity.

• Chromosome 1 has the most genes ( 2968) and the Y chromosome has the fewest (231).

• Unlike the human’s seemingly random distribution of gene rich areas, many other organisms genomes are more uniform with genes evenly spaced throughout.

• Humans have on an average three times as many kinds of proteins as the fly and the worm. This is because of mRNA transcript’s alternate splicing and chemical modifications to the proteins. This process can yield different protein products from the same gene.

• Humans share most of the same protein families with worms, flies and plants. However, the number of gene family members is expanded in humans especially in proteins involved in development and immunity.

• Human genome has much greater portion of repeat sequences (50%) than Arabidopsis (mustard weed) (11%), the worm (7%), and the fly (3%).

• Although humans appear to have stopped accumulating repeated DNA sequences over 50 million years ago, there seems to be no such decline in rodents. This may account for some of the fundamental differences between hominids and rodents. Although gene estimates are similar in these species. Scientists have proposed many theories to explain evolutionary contrasts between humans and other organisms including those of life span, litter sizes, inbreeding, genetic drift etc.

• In 2003, fine sequences have been submitted.

• As per latest estimate, now total 24847 genes have been predicted in the entire human genome.

VARIATIONS AND MUTATIONS

• About 1.4 million locations have been identified where single base differences occur in humans. This information promises to revolutionize the processes of finding chromosomal locations for disease associated sequences and tracing human history.

• The human sequence project made the biologists acquainted with the importance of single nucleotide polymorphism (SNP). The SNP’s define differences among people, predispose a person to disease, and influence a patient’s response to a drug. Now, it is possible to build microarrays that can target individual SNP variations, as well as making deeper comparisons across the genome.

• The ratio of germline ( sperm or egg cell ) mutations is 2:1 in males versus females. Researchers give several reasons for the higher mutation rate in the male germline including the greater number of cell divisions required for sperm formation than for eggs.

GENE PREDICTIONS

Organism Size (Mb) Year of sequence

No. of genes Gene density

Saccharomyces cerevisiae

12.1 1996 6034 483

Escherichia coli 4.6 1997 4200 932

Caenorhabditis elegans

(roundworm)

97 1998 19099 197

Arabidopsis thaliana

100 2000 25000 221

Drosophila melanogaster

180 2000 13061 117

Human 3200 2001 25000 12

• Gene predictions are made by computational algorithms based on recognition of gene sequence features and similarities to known genes. Gene estimates need further confirmation including characterization of their protein products and functions.

• Gene density = Number of genes per million sequenced DNA bases.

STRATEGIES FOR SEQUENCING THE HUMAN GENOME

• The strategy originally established by the publicly funded effort (HGP) was based on localizing bacterial artificial chromosomes (BACs) containing large fragments of human DNA within the framework of a landmark-based physical map.

• Ideally sequencing would have been done on a clone- by- clone basis, with clones selected from the minimum BAC tiling path (i.e, a set of BACs that, with minimum overlap, stretched across the whole length of the genome).

• The working draft, although containing some gaps and ambiguities in order, will be extremely useful in such efforts as identifying disease-associated genes.

• The idealized strategy of celera was to avoid the up-front mapping phase by subcloning random fragments of the human genome directly.

• Sequencing of both ends of fragments in libraries of different sizes facilitated ordering.

• While saving effort and time at the beginning, the Celera approach would make the assembly process much more dependent on algorithms and computer time.

• In their efforts, to reach their goals, the idealized strategies evolved into hybrids in which the HGP selected more clones arbitrarily and Celera made use of BAC maps and sequence generated by HGP.

DO IT YOURSELF SCIENCE

• With the sharply falling costs of equipment and wealth of information that is publicly available, we are getting to the point at which almost anyone with access to the internet and equipment for sequencing can publish his/her genetic information.

• It is evident from the recent story published in Nature about Hugh Rienhoff, a trained geneticist and biotechnology entrepreneur, whose daughter was born with a collection of congenital defects.

• He investigated the genetic cause by himself by buying lab equipments and having her gene sequenced.

• He posted his theories behind the possible cause of disease and posted information about her condition and genetic sequence on the internet.

• Besides, the recent release of greatly enhanced haplotype map or HapMap, describes the most common forms of human genetic variation characterizing over 3.1 million human SNPs across geographically diverse populations.

• These findings demonstrate the power of genomics to deliver clues that could yield better medicine and uncovering multiple genes that may be associated with the risk of developing specific diseases.

APPLICATIONS, FUTURE CHALLENGES

• Deriving meaningful knowledge from the DNA sequence will define research to inform understanding of biological systems. This task will require expertise and creativity of tens of thousands of scientists from varied disciplines in both the public and private sectors worldwide.

• Having this sequence will enable the workers a new approach to biological research. In the past, researchers studied one or few genes at a time. With whole genome sequences and new high throughput technologies, they can approach questions systematically and on a grand scale. They can study all the genes in a genome, for example, or all the transcripts in a particular tissue or organ or tumor, or how tens of thousands of genes and protein work together in interconnected networks to orchestrate the chemistry of life.

ACKNOWLEDGEMENTS

For this talk, the matter has been collected from Human Genome News published by the US Department of Energy Office of biological and environmental research ( July 2001 issue).