The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

1. Introduction Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1–4. Primer3 is a computer program that suggests PCR primers for a variety of applications, for example to create STSs (sequence tagged sites) for radiation hybrid mapping (5), or to amplify sequences for single nucleotide polymor-phism discovery (6). Primer3 can also select single primers for sequencing reactions and can design oligonucleotide hybridization probes. In selecting oligos for primers or hybridization probes, Primer3 can consider many factors. These include oligo melting temperature, length, GC content , 3′ stability, estimated secondary structure, the likelihood of annealing to or amplifying undesirable sequences (for example interspersed repeats), the likelihood of primer–dimer formation between two copies of the same primer, and the accuracy of the source sequence. In the design of primer pairs Primer3 can consider product size and melting temperature, the likelihood of primer– dimer formation between the two primers in the pair, the difference between primer melting temperatures, and primer location relative to particular regions of interest or to be avoided.

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Primer3 on the WWW for general users and for biologist programmers.

As vertebrate genome sequences near completion and research refocuses to their analysis, the issue of effective genome annotation display becomes critical. A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided at http://genome.ucsc.edu. This browser displays assembly contigs and gaps, mRNA and expressed sequence tag alignments, multiple gene predictions, cross-species homologies, single nucleotide polymorphisms, sequence-tagged sites, radiation hybrid data, transposon repeats, and more as a stack of coregistered tracks. Text and sequence-based searches provide quick and precise access to any region of specific interest. Secondary links from individual features lead to sequence details and supplementary off-site databases. One-half of the annotation tracks are computed at the University of California, Santa Cruz from publicly available sequence data; collaborators worldwide provide the rest. Users can stably add their own custom tracks to the browser for educational or research purposes. The conceptual and technical framework of the browser, its underlying MYSQL database, and overall use are described. The web site currently serves over 50,000 pages per day to over 3000 different users.

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The Human Genome Browser at UCSC

The character of a cell is defined by its constituent proteins, which are the result of specific patterns of gene expression. Crucial determinants of gene expression patterns are DNA-binding transcription factors that choose genes for transcriptional activation or repression by recognizing the sequence of DNA bases in their promoter regions. Interaction of these factors with their cognate sequences triggers a chain of events, often involving changes in the structure of chromatin, that leads to the assembly of an active transcription complex (e.g., Cosma et al. 1999). But the types of transcription factors present in a cell are not alone sufficient to define its spectrum of gene activity, as the transcriptional potential of a genome can become restricted in a stable manner during development. The constraints imposed by developmental history probably account for the very low efficiency of cloning animals from the nuclei of differentiated cells (Rideout et al. 2001; Wakayama and Yanagimachi 2001). A “transcription factors only” model would predict that the gene expression pattern of a differentiated nucleus would be completely reversible upon exposure to a new spectrum of factors. Although many aspects of expression can be reprogrammed in this way (Gurdon 1999), some marks of differentiation are evidently so stable that immersion in an alien cytoplasm cannot erase the memory. The genomic sequence of a differentiated cell is thought to be identical in most cases to that of the zygote from which it is descended (mammalian B and T cells being an obvious exception). This means that the marks of developmental history are unlikely to be caused by widespread somatic mutation. Processes less irrevocable than mutation fall under the umbrella term “epigenetic” mechanisms. A current definition of epigenetics is: “The study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” (Russo et al. 1996). There are two epigenetic systems that affect animal development and fulfill the criterion of heritability: DNA methylation and the Polycomb-trithorax group (Pc-G/trx) protein complexes. (Histone modification has some attributes of an epigenetic process, but the issue of heritability has yet to be resolved.) This review concerns DNA methylation, focusing on the generation, inheritance, and biological significance of genomic methylation patterns in the development of mammals. Data will be discussed favoring the notion that DNA methylation may only affect genes that are already silenced by other mechanisms in the embryo. Embryonic transcription, on the other hand, may cause the exclusion of the DNA methylation machinery. The heritability of methylation states and the secondary nature of the decision to invite or exclude methylation support the idea that DNA methylation is adapted for a specific cellular memory function in development. Indeed, the possibility will be discussed that DNA methylation and Pc-G/trx may represent alternative systems of epigenetic memory that have been interchanged over evolutionary time. Animal DNA methylation has been the subject of several recent reviews (Bird and Wolffe 1999; Bestor 2000; Hsieh 2000; Costello and Plass 2001; Jones and Takai 2001). For recent reviews of plant and fungal DNA methylation, see Finnegan et al. (2000), Martienssen and Colot (2001), and Matzke et al. (2001).

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DNA methylation patterns and epigenetic memory

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.

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Initial sequencing and comparative analysis of the mouse genome.

Interspersed repeats and other mementos of transposable elements in mammalian genomes.

The origin of interspersed repeats in the human genome.

Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel.

We report eleven new families of MEdium Reiteration frequency (MER) interspersed repeats in the genomes of Primates, Rodentia, and Lagomorpha. Two families of the human repeats, MER46 and MER47, represent non-autonomous DNA transposons. These sequences are flanked by TA target site duplications and have terminal inverted repeats (TIRs) similar to TIRs of DNA transposons. The sequences of five other families of repeats, MER41, MER48, MER50, MER51, and RMER3, resemble long terminal repeats of retroviruses. A potential involvement of some of the reported MER repeats in the regulation of transcription and genetic rearrangements is suggested. Age estimations place the origin of most MER repeats at the time of decline in MIR (Mammalian-wide Interspersed Repeats) retroposition and before the origin of the Alu family.

Identification of new medium reiteration frequency repeats in the genomes of Primates, Rodentia and Lagomorpha

The prion protein (PrP), first identified in scrapie-infected rodents, is encoded by a single-copy chromosomal gene. PrP genes are conserved and have been identified in 13 species of mammals. Cloning and analysis of the PrP gene has revealed that pathogenic (PrPSc) and cellular (PrPC) isoforms of the prion protein do not reflect alternative splicing events but instead share the same amino acid sequence: these data first suggested the hypothesis that prion isoforms are alternate conformers1. However, at this time the mechanism involved in the transition between PrPC and PrPSc is obscure, as is the normal function of PrPC. We have therefore sought to identify PrP-linked genes that might feature in conformational transitions, and conserved regulatory elements that might offer insights into the physiology of PrPC. Accordingly, 145 kb of DNA from human and sheep PrP genes, and two alleles of Prn-p has been analysed for conserved regions, ORFs, potential exons, repetitive sequences, putative CpG islands, and possible polymorphic motifs. These sequences reveal unexpected features within the wild-type PrP genes of each of these species. The predominant allele of the mouse gene, Prn-pa, found in 44 inbred laboratory strains contains a 6878 nucleotide retroviral genome inserted into the anticoding strand of intron 2. This intracisternal A particle (IAP) element is (i) flanked by duplications of a AAGCTT nucleotide motif found once in the Prn-pb gene, and (ii) highly related to a transpositionally-competent prototype IAP element, differing principally in small deletion of the pol gene: these data are indicative of a recent transpositional orgin. In the case of the sheep PrP gene, the unusually long 3′ untranslated region (UTR) reflects in most part the presence of a “fossil” 1.2 kb mariner-like transposable element, which is absent from the human and mouse PrP genes. Chromosomal instability associated with mariner-like elements on chromosome 17 in humans suggests that DNA rearrangements may take place adjacent to the PrP gene in sheep, and also in cattle (which also exhibit an extended 3′ UTR and presumably harbor a mariner-like element). Lastly, the large intron of the human PrP gene contains a sequence analogous to exon 2 of the mouse and sheep PrP genes, flanked by consensus splice acceptor and donor sites: while it remains to be established that “exon 2” sequences are included in a subset of human PrP mRNAs, these data indicate that an ancestral PrP gene most likely exhibited a three exon structure.

Arian Fa Smit

Papers

Interspersed repeats and other mementos of transposable elements in mammalian genomes.

The origin of interspersed repeats in the human genome.

Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel.

Identification of new medium reiteration frequency repeats in the genomes of Primates, Rodentia and Lagomorpha

Large-Scale Sequencing of Human, Mouse, and Sheep Prion Protein Genes