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.

We describe a new basis for the construction of a genetic linkage map of the human genome. The basic principle of the mapping scheme is to develop, by recombinant DNA techniques, random single-copy DNA probes capable of detecting DNA sequence polymorphisms, when hybridized to restriction digests of an individual's DNA. Each of these probes will define a locus. Loci can be expanded or contracted to include more or less polymorphism by further application of recombinant DNA technology. Suitably polymorphic loci can be tested for linkage relationships in human pedigrees by established methods; and loci can be arranged into linkage groups to form a true genetic map of "DNA marker loci." Pedigrees in which inherited traits are known to be segregating can then be analyzed, making possible the mapping of the gene(s) responsible for the trait with respect to the DNA marker loci, without requiring direct access to a specified gene's DNA. For inherited diseases mapped in this way, linked DNA marker loci can be used predictively for genetic counseling.

Construction of a genetic linkage map in man using restriction fragment length polymorphisms.

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

PROBLEM TO BE SOLVED: To provide a method for preparing a short nucleotide sequence (tag) which is useful to identify a cDNA oligonucleotide and is derived from a restricted position in a mRNA or a cDNA. SOLUTION: This is the method of preparing a tag for identifying the cDNA oligonucleotide. The above method comprises preparing the cDNA oligonucleotide bearing 5' and 3' terminals, collecting cDNA fragments by cutting the cDNA oligonucleotide with a restriction enzyme at the first restriction endonuclease site, separating a cDNA oligonucleotide bearing 5' or 3' terminal and connecting an oligonucleotide linker to the isolated cDNA fragment bearing the cDNA oligonucleotide 5' or 3' terminal. Here, the oligonucleotide linker contains the recognition site of the second restriction endonuclease enzyme and the isolated cDNA fragment is cut with the second restriction endonuclease enzyme which cuts the cDNA fragment in a section separated from the recognition site to obtain the tag for identifying the cDNA oligonucleotide.

http://science.sciencemag.org/content/sci/270/5235/484.full.pdf

Serial analysis of gene expression

Maturation of the head of bacteriophage T4. I. DNA packaging events.

The technical problem of detecting the retrotransposition—amplification and genomic dispersion of sequences through an RNA intermediate—of Pol III–directed transcripts has been solved. This provides a long-sought experimental system to learn how Alu repeats amplified to a high copy number in human DNA.

Alu: a parasite's parasite?

A dimer satellite sequence in bonnet monkey DNA consists of distinct monomer subunits.

The transcriptional activity of a human transposon-like family of repeats, called the THE-1 family, has been studied in cell culture and in human tissue. Both strands of THE-1 are present in several discrete length poly A plus RNAs. Primer extension studies and the structures of cDNA clones show that these THE-1 transcripts are usually the product of other transcription units. The THE-1 LTR provides the polyadenylation processing site for two transcripts, which result from upstream non THE-1 promoters. Yet another transcript, containing an internal THE-1 element in the probable sense orientation, is greatly enriched in a polysomal size fraction.

/pdf/transcription-of-a-human-transposon-like-sequence-is-usually-4frmhhph7m.pdf

Transcription of a human transposon-like sequence is usually directed by other promoters.

Publisher Summary This chapter describes the evolutionary analyses of repetitive DNA sequences. The study of repetitive DNA sequences is refined by advances in cloning, sequencing, and oligonucleotide synthesis. Whereas the original studies of repeated DNA evolution analyzes whole families of repeated DNA sequences using genomic hybridization techniques, it is now possible to use cloning and DNA sequence analysis to define subfamilies of repeated DNA sequences. These subfamilies may then be characterized rapidly utilizing other approaches, such as specific oligonucleotide probes and the polymerase chain reaction (PCR). It is difficult to improve on direct DNA sequence comparisons for evolution studies of the repeated DNA sequences. The only drawback is that these studies are relatively labor intensive, limiting the experimental sample to a much smaller one than can be studied using hybridization procedures. The chapter highlights the special considerations for repetitive DNA evolution.

https://biosci-batzerlab.biology.lsu.edu/Publications/Batzer_MethEnzy_1993.pdf

Evolutionary analyses of repetitive DNA sequences.

The organization of U2 genes was compared in apes, Old World monkeys, and the prosimian galago. In humans and all apes (gibbon, orangutan, gorilla, and chimpanzee), the U2 genes were organized as a tandem repeat of a 6-kb element; however, the restriction maps of the 6-kb elements in these divergent species differed slightly, demonstrating that mechanisms must exist for maintaining sequence homogeneity within this tandem array. In Old World monkeys, the U2 genes were organized as a tandem repeat of an 11-kb element; the restriction maps of the 11-kb elements in baboon and two closely related macaques, bonnet and rhesus monkeys, also differed slightly, confirming that efficient sequence homogenization is an intrinsic property of the U2 tandem array. Interestingly, the 11-kb monkey repeat unit differed from the 6-kb hominid repeat unit by a 5-kb block of monkey-specific sequence. Finally, we found that the U2 genes of the prosimian galago were dispersed rather than tandemly repeated, suggesting that the hominid and Old World monkey U2 tandem arrays resulted from independent amplifications of a common ancestral U2 gene. Alternatively, the 5-kb monkey-specific sequence could have been inserted into the 6-kb array or deleted from the 11-kb array soon after divergence of the hominid and Old World monkey lineages.

Carl W. Schmid

Papers

Alu: a parasite's parasite?

A dimer satellite sequence in bonnet monkey DNA consists of distinct monomer subunits.

Transcription of a human transposon-like sequence is usually directed by other promoters.

Evolutionary analyses of repetitive DNA sequences.

Structure and evolution of the U2 small nuclear RNA multigene family in primates: gene amplification under natural selection?