Sequence Of Human Chromosome Research Articles

Private Science: Biotechnology and the Rise of the Molecular Sciences (review)

When Edward R. Murrow declared in 1964, "the speed of communications is wondrous to behold," 1 he could not have envisioned the even faster-moving future of biotechnology. Originating from a 1993 Chemical Heritage Foundation conference, this volume strives (as editor and CHF director Arnold Thackray notes in his introduction) to make an original "contribution to the new discourse on the structure and significance of biotechnology and the Biomolecular Revolution" (p. viii). But in the eighteen months since its press time, pundits of biotechnology have seen even their most up-to-date conclusions challenged by such "wondrous" developments as the first complete human chromosome sequence, the first gene-therapy death, and the first large-scale American public demonstrations against the genetically altered crops that dominate U.S. agriculture. So this whole enterprise begs some important historical questions: What constitutes a scientific revolution-in-progress? And how do its contemporaneous analysts best chronicle and understand it, especially when it is developing and changing so quickly?

Genomic structure of a copy of the human TPTE gene which encompasses 87 kb on the short arm of chromosome 21.

The testis-expressed human TPTE is a putative transmembrane tyrosine phosphatase, probably involved in signal transduction pathways of the endocrine and/or the spermatogenetic function of the testis. TPTE was mapped to the pericentromeric region of human chromosomes 21 and 13, and to chromosomes 15, 22, and Y. It is unknown which of the TPTE copies are transcribed, contain intronic sequences, and/or have open reading frames. Here, in silico analysis of the genomic sequence of human chromosome 21 allowed the determination of the genomic structure of a copy of the TPTE gene. This copy consists of 24 exons and spans approximately 87 kb. The mapping position of this copy of TPTE on the short arm of chromosome 21 was confirmed by FISH using the BAC 15L0C0 clone as a probe that contains almost the entire TPTE gene. This is the first description of the genomic sequence of a non-RNR gene on the short arm of human acrocentric chromosomes.

Down to less than 300 genes: the DNA sequence of human chromosome 21 and Down’s syndrome

The human chromosome 21 has been sequenced1xThe DNA sequence of human chromosome 21. Hattori, M. et al. Nature. 2000; 405: 311–319Crossref | PubMed | Scopus (776)See all References1, a major step in our quest for decoding the blue print of human life. The sequence data covers 99.7% of 21q, the long arm of the chromosome, as well as a portion of 21p, the short arm, and is estimated to be 99.995% accurate. Surprisingly only 225 genes and 59 pseudogenes (genes that are incapable of being expressed as protein products) were identified. Although these estimates may only be considered approximate, they certainly suggest that chromosome 21, and thus perhaps the entire human genome too, contains far fewer genes than was previously thought.More than 43% of the detected genes are novel and 41% of them have no functional attributes. Some functionally related gene clusters (e.g. the interferon receptor family, or the trefoil peptide cluster) were found to be arranged in tandem arrays, suggesting the possibility of rounds of gene duplication during evolution. The sequence also confirmed that compositional features of the chromosome (e.g. guanine and cytosine content, or the presence of Alu repeats) correlate well with gene density. Furthermore, several duplicated regions were detected on the chromosome. The function of these regions is not understood but might be relevant for mechanisms involved in chromosomal rearrangements.The sequencing of chromosome 21 will have significant medical implications: several of its genes have been implicated in a number of human diseases and perhaps one of the most important of these is Down’s syndrome, a disease that affects one in 700 live births. The cause of Down’s syndrome, the duplication of some or all of the, 21st chromosome (leading to three, instead of two copies of genes) has long been known. Nevertheless, the problem of which genes are responsible for the characteristic abnormalities of this disease, ranging from mental retardation to heart problems, has remained elusive. It was suspected that the complex phenotypical alterations of Down’s syndrome could be the result of an interplay between several genes. However, without knowing all potentially relevant genes it was difficult to dissect the mechanisms of the disease. With the sequencing of chromosome 21 this obstacle has now been greatly diminished: for the first time, it has become possible to systematically investigate the role of every resident gene. Understanding the genetic mechanisms of Down’s syndrome will be an enormous scientific achievement in itself. But by doing so, we might also be holding the key to another devastating neurodegenerative disease, Alzheimer’s Disease, which is prevalent in Down’s patients of 35 years or older but which also affects a large proportion of the elderly in the general human population. Clearly, the medical importance of knowing the sequence of the 21st chromosome cannot be overestimated.However, the sequence is only a series of characters, a book written in a secret language whose message we will yet need to understand by translating it to the language of biological function. With the pieces in hand we can now start putting the puzzle together, which will eventually reveal the picture we have long been seeking.

Genome sequencing: mind the gap.

The year 2000 is poised to be declared the ‘Year of the Genome’. The sequences of human chromosomes 21 and 22 have been published in the past six months, and two further landmark announcements are expected within days — indeed, may even have been made by the time you read this. Scientists of the Human Genome Project are on the verge of announcing completion of a “rough draft” of the human genome. And the world should soon hear another stunning proclamation from Celera Genomics, which recently completed a genome draft and is now assembling its data into near-final form.

The DNA sequence of human chromosome 21.

Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

Erratum: correction: The DNA sequence of human chromosome 22

Nature 402, 489–495 (1999) The names of the following authors were omitted from the complete list at the end of the Article: P. Wilkinson (The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK); A. Bodenteich, K. Hartman, X. Hu, A. S. Khan, L. Lane, Y. Tilahun & H. Wright (Department of Chemistry and Biochemistry, The University of Oklahoma, 620 Parrington Oval, Room 311, Norman, Oklahoma 73019, USA).

Autonomous Distributed Genome Database and Its Application to Mouse cDNA and Human Genome Sequence Matching

The vast increase of genomic sequence data reveals the limitation of single computer systems in terms of both processing and storage capacity. The only realistic way to cope with this growth in the near future will consist in using several computers rather than a single one. Catching up with the steady growth of genomic sequence data is likely to require systems with an increasingly large number of nodes. For this purpose, solutions based on PC clusters are attractive because they offer good performance for the price, and provide reasonable hardware scalability. Nevertheless, building such a system raises at least three important concerns: (1) the whole system, including the database management, must be scalable and accommodate a very large number of server nodes (say, tens of thousands), (2) the system must tolerate the failure individual nodes because this happen frequently as their number increases, and (3) the whole system should appear to clients as a single database image. Considering this context, we sketch the overall architecture of an autonomous scalable genome database which should satisfy the requirements mentioned above. The system consists of partially independent databases, with a management system to provides a single database image. Our prototype system makes use of XML to store information, and of Java servlet/applet technology for autonomous data management and remote access. As an illustration, we have tested on BLAST search results between mouse cDNA and human chromosome sequence.

The sequence of human chromosome 21 and implications for research into Down syndrome.

The recent completion of the DNA sequence of human chromosome 21 has provided the first look at the 225 genes that are candidates for involvement in Down syndrome (trisomy 21). A broad functional classification of these genes, their expression data and evolutionary conservation, and comparison with the gene content of the major mouse models of Down syndrome, suggest how the chromosome sequence may help in understanding the complex Down syndrome phenotype.

Sequence of human chromosome 21

Sequence of human chromosome 21

The DNA sequence of human chromosome 22.

Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

Evolution of the centromeric alpha-satellite DNA sequences of human chromosome 22

Evolution of the centromeric alpha-satellite DNA sequences of human chromosome 22

A Contiguous 3-Mb Sequence-Ready Map in the S3–MX Region on 21q22.2 Based on High- Throughput Nonisotopic Library Screenings

Progress in complete genomic sequencing of human chromosome 21 relies on the construction of high-quality bacterial clone maps spanning large chromosomal regions. To achieve this goal, we have applied a strategy based on nonradioactive hybridizations to contig building. A contiguous sequence-ready map was constructed in the Down syndrome congenital heart disease (DS-CHD) region in 21q22.2, as a framework for large-scale genomic sequencing and positional candidate gene approach. Contig assembly was performed essentially by high throughput nonisotopic screenings of genomic libraries, prior to clone validation by (1) restriction digest fingerprinting, (2) STS analysis, (3) Southern hybridizations, and (4) FISH analysis. The contig contains a total of 50 STSs, of which 13 were newly isolated. A minimum tiling path (MTP) was subsequently defined that consists of 20 PACs, 2 BACs, and 5 cosmids covering 3 Mb between D21S3 and MX1. Gene distribution in the region includes 9 known genes (c21-LRP, WRB, SH3BGR, HMG14, PCP4, DSCAM, MX2, MX1, and TMPRSS2) and 14 new additional gene signatures consisting of cDNA selection products and ESTs. Forthcoming genomic sequence information will unravel the structural organization of potential candidate genes involved in specific features of Down syndrome pathogenesis.

12] Chromosome spread for confocal microscopy

The chapter describes the use of confocal laser scanning microscopy (CLSM) in the detection of specific DNA sequences in human chromosomes. The chapter discusses the methods of chromosome spreading that allow the maintenance of a suitable chromosome structure and to the labeling procedures utilized for fluorescence and reflectance CLSM identification of specific chromosome probes. CLSM provides improved lateral and axial resolution; moreover, the series of optical sections can be recombined digitally to render three-dimensional (3-D) reconstruction. CLSM can combine fluorescence with reflectance signals, allowing the combination of fluorochromes and colloidal gold labeling, thus providing resolution performances very close to the theoretical limit of the system. The main prerequisites for successful utilization of CLSM in chromosome analysis are as follows: (1) preparation of high-quality chromosome spreads in which contaminant proteins are removed completely and chromosome ultrastructural organization is maintained and (2) setting of the CLSM when different signals are collected to maintain lateral and axial resolution without signal displacement.

Solution structure of a Na cation stabilized DNA quadruplex containing G·G·G·G and G·C·G·C tetrads formed by G-G-G-C repeats observed in adeno-associated viral DNA

We have applied NMR and molecular dynamics computations including intensity based refinement to define the structure of the d(G-G-G-C-T4-G-G-G-C) dodecanucleotide in 100 mM NaCl solution. The G-G-G-C sequence is of interest since it has been found as tandem repeats in the DNA sequence of human chromosome 19. The same G-G-G-C sequence is also seen as islands in adeno-associated virus, a human parvovirus, which is unique amongst eukaryotic DNA viruses in its ability to integrate site-specifically into a defined region of human chromosome 19. The d(G-G-G-C-T4-G-G-G-C) sequence forms a quadruplex in Na cation containing solution through head-to-tail dimerization of two symmetry-related stem-hairpin loops with adjacent strands antiparallel to each other around the quadruplex. The connecting T4 loops are of the lateral type, resulting in a quadruplex structure containing two internal G·G·G·G tetrads flanked by G·C·G·C tetrads. The G(anti)·G(syn)·G(anti)·G(syn) tetrads are formed through dimerization associated hydrogen bonding alignments of a pair of Hoogsteen G(anti)·G(syn) mismatch pairs, while the G(anti)·C(anti)·G(anti)·C(anti) tetrads are formed through dimerization associated bifurcated hydrogen bonding alignments involving the major groove edges of a pair of Watson-Crick G·C base-pairs. The quadruplex contains two distinct narrow and two symmetric wide grooves with extensive stacking between adjacent tetrad planes. The structure of the quadruplex contains internal cavities that can potentially accommodate Na cations positioned between adjacent tetrad planes. Three such Na cations have been modeled into the structure of the d(G-G-G-C-T4-G-G-G-C) quadruplex. Finally, we speculate on the potential role of quadruplex formation involving G·G·G·G and G·C·G·C tetrads during the integration of the adeno-associated parvovirus into its target on human chromosome 19, both of which involve stretches of G-G-G-C sequence elements.

Human gene targeting by viral vectors.

Stable transduction of mammalian cells typically involves random integration of viral vectors by non-homologous recombination. Here we report that vectors based on adeno-associated virus (AAV) can efficiently modify homologous human chromosomal target sequences. Both integrated neomycin phosphotransferase genes and the hypoxanthine phosphoribosyltransferase gene were targeted by AAV vectors. Site-specific genetic modifications could be introduced into approximately 1% of cells, with the highest targeting rates occurring in normal human fibroblasts. These results suggest that AAV vectors could be used to introduce specific genetic changes into the genomic DNA of a wide variety of mammalian cells, including therapeutic gene targeting applications.

Padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21.

Chromosome centromeres, composed of repeated DNA sequences, orchestrate the correct segregation of chromatids in cell division. We have examined the centromeres of human chromosomes 13 and 21 by studying the distribution, in situ, of two alpha satellite sequences that differ in a single nucleotide position. This was possible using padlock probes, oligo-nucleotides that can be ligated into circles upon target recognition. The segregation of individual 13 and 21 homologues in a family was followed by monitoring of the signals from two differentially labelled probes, specific for either sequence variant. A characteristic arrangement of the repeat motifs in three separate spots, oriented transverse to the length axis of the metaphase chromosomes and bilaterally symmetric, indicates that only parts of the detected regions are involved in the centromeric region, joining the sister chromatids before anaphase.

Human pseudoautosomal boundary-like sequences: expression and involvement in evolutionary formation of the present-day pseudoautosomal boundary of human sex chromosomes.

The human genome is composed of long-range mosaic structures of G+C% (GC%), which are thought to be related to chromosome bands. We previously identified a boundary of Mb-level domains of GC% mosaic structures in the human major histocompatibility complex (MHC) and found in the domain boundary a sequence very similar to pseudoautosomal boundary (PAB) sequences of human sex chromosomes. We designated it 'PABL' and found many PABLs in the human genome. By analysis of six genomic and six transcribed PABLs, a core and consensus sequence of about 650 nt was defined; the 3'- and 5'-edges of the PABLs were strictly conserved. Northern blot analysis showed sizes of PABL transcripts to be 5-10 kb in length. Divergence time of PABLs was estimated to be 60-120 million years ago by analysis of human PABLs and PABXY1 of seven primates, and the evolutionary rates deduced showed PABLs to have been under selective constraints. A model for evolutionary formation of the present pseudoautosomal boundary was proposed by postulation of illegitimate recombination between two PABLs.

An arrayed library enriched in hncDNA corresponding to transcribed sequences of human chromosome 19: preparation and analysis

A simple technique for preparation of libraries of the human chromosome specific transcribed sequences is developed. It uses hnRNA from human-rodent hybrid cell lines containing particular human chromosomes or their fragments and includes three stages: (i) reverse transcription of the hnRNA with Alu-specific primers directing the transcription beyond the Alu-repeats to flanking non-repetitive sequences of the chromosome; (ii) nested primer PCR strategy with specifically designed primers; (iii) direct selective cloning of the second-stage nested primer PCR products. An arrayed hncDNA library was prepared from a hybrid cell line containing chromosome 19 and fragments of 22 and X chromosomes. The library contains aorund 98% of human-specific transcribed sequences. Sequences of 52 human-specific, according to PCR analysis, clones differed from each other and had no close analogs in the EMBL Data Bank. Of 17 clones assigned to certain human chromosomes, 9 belonged to chromosome 19, 5 to chromosome 22 and 3 to chromosome X. Some of the human specific clones contained repetitive elements scattered over different human chromosomes. Clones from hncDNA libraries are useful as STSs/ESTs, as probes for detecting full-size genes in genomic libraries, for RFLP analysis and for identification of chromosome specific cDNAs.

A method for the rapid generation of alpha- and classical satellite probes for human chromosome 9 by polymerase chain reaction using genomic DNA and their application to detect chromosomal alterations in interphase cells.

Fluorescence in situ hybridization (FISH) using chromosome-specific DNA probes is a technique which has recently become widely used for the analysis of chromosome alterations in interphase and metaphase cells. In this report, a polymerase chain reaction (PCR)-based method is described for simultaneously amplifying and labelling probes targeting the alpha- and classical satellite regions of chromosome 9 using either plasmid or genomic DNA. Chromosome-specific probes were generated using readily obtainable plasmid DNA and genomic DNA from a hybrid cell line containing human chromosome 9 in a hamster cell background. The utility of these probes to detect and quantify structural and numerical aberrations in interphase cells was demonstrated using a new multicolor FISH strategy by comparing the frequencies of hyperdiploidy and chromosome breakage affecting the regions targeted by the probes in interphase and metaphase human lymphocytes irradiated during culture. The irradiated cells exhibited a significantly higher frequency of tetrasomy and breakage effecting the centromeric/pericentric region of chromosome 9 as compared with non-exposed cells. In general, similar frequencies of breakage and hyperdiploidy were observed in the interphase and metaphase preparations. These results show that DNA probes for the repetitive sequences in human chromosomes can be easily generated from genomic DNA and that these probes can be effectively used to detect chromosome breakage and aneuploidy in interphase and metaphase lymphocytes in vitro.

Construction of a panel of chromosome-specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ.

We present the sequences of a set of oligonucleotides that, when used as primers for PRimed IN Situ (PRINS) labeling, are diagnostic for repetitive sequences in specific human chromosomes. Combined, they enable identification of all human chromosomes except 6, 19, and 20. However, as is also the case with cloned centromeric hybridization probes, chromosomes 14 and 22 are stained together. Along with the sequences of these oligonucleotides we offer a simple, universal procedures for their use. We also present an oligonucleotide that binds to both strands of alpha-satellite DNA, making it possible to specifically amplify alpha-satellite DNA by PCR with this one oligonucleotide alone as primer. The origin of the alpha-satellite DNA in the starting material (whether somatic cell hybrids, flow-sorted chromosomes, or microdissected material) can then be determined on test metaphase spreads by in situ hybridization or PRINS with the PCR product.