A physical map of the human Y chromosome

The non-recombining region of the human Y chromosome (NRY), which comprises 95% of the chromosome, does not undergo sexual recombination and is present only in males. An understanding of its biological functions has begun to emerge from DNA studies of individuals with partial Y chromosomes, coupled with molecular characterization of genes implicated in gonadal sex reversal, Turner syndrome, graft rejection and spermatogenic failure. But mapping strategies applied successfully elsewhere in the genome have faltered in the NRY, where there is no meiotic recombination map and intrachromosomal repetitive sequences are abundant. Here we report a high-resolution physical map of the euchromatic, centromeric and heterochromatic regions of the NRY and its construction by unusual methods, including genomic clone subtraction and dissection of sequence family variants. Of the map's 758 DNA markers, 136 have multiple locations in the NRY, reflecting its unusually repetitive sequence composition. The markers anchor 1,038 bacterial artificial chromosome clones, 199 of which form a tiling path for sequencing.     

Integration of telomere sequences with the draft human genome sequence

Telomeres are the ends of linear eukaryotic chromosomes. To ensure that no large stretches of uncharacterized DNA remain between the ends of the human working draft sequence and the ends of each chromosome, we would need to connect the sequences of the telomeres to the working draft sequence. But telomeres have an unusual DNA sequence composition and organization that makes them particularly difficult to isolate and analyse. Here we use specialized linear yeast artificial chromosome clones, each carrying a large telomere-terminal fragment of human DNA, to integrate most human telomeres with the working draft sequence. Subtelomeric sequence structure appears to vary widely, mainly as a result of large differences in subtelomeric repeat sequence abundance and organization at individual telomeres. Many subtelomeric regions appear to be gene-rich, matching both known and unknown expressed genes. This indicates that human subtelomeric regions are not simply buffers of nonfunctional 'junk DNA' next to the molecular telomere, but are instead functional parts of the expressed genome.     

Suppression of tumorigenicity in human colon carcinoma cells by introduction of normal chromosome 5 or 18.

Development of colon carcinomas can be associated with allelic deletions on several chromosomes, including 5q and 18q. The APC gene on 5q and the DCC gene on 18q have been identified as potential tumour suppressor genes, whose suppression contributes to colon carcinogenesis. To investigate the role of genes in these deleted regions, we have now introduced a single normal human chromosome into a human colon carcinoma cell line, COKFu, through microcell hybridization. Several clones of hybrid cells containing normal chromosome 5, and others containing normal chromosome 18, were obtained. The morphology of the hybrid cells was markedly altered: the hybrids with chromosome 5 exhibited a closely packed polygonal morphology, and the hybrid cells with chromosome 18 were flattened. The cloning efficiency of the hybrid cells in soft agar was reduced from 0.46 to 0% of that of the parental carcinoma cells, and the tumorigenicity of these hybrid cells in athymic nude mice was completely suppressed. The growth properties of the hybrid cells with chromosome 11 were not substantially changed. These results strongly suggest that the genes on normal chromosome 5 and 18 function as tumour suppressors in colon carcinogenesis.     

Genome linking with yeast artificial chromosomes

The haploid genome of Caenorhabditis elegans consists of some 80 x 10(6) base pairs of DNA contained in six chromosomes. The large number of interesting loci that have been recognized by mutation, and the accuracy of the genetic map, mean that a physical map of the genome is highly desirable, because it will facilitate the molecular cloning of chosen loci. The first steps towards such a map used a fingerprinting method to link cosmid clones together. This approach reached its practical limit last year, when 90-95% of the genome had been cloned into 17,500 cosmids assembled into some 700 clusters (contigs), but the linking clones needed were either non-existent or extremely rare. Anticipating this, we had planned to link by physical means--probably by hybridization to NotI fragments separated by pulse field gel electrophoresis. NotI recognizes an eight base sequence of GC pairs; thus the fragments should be large enough to bridge regions that clone poorly in cosmids, and, with no selective step involved, would necessarily be fully representative. However, with the availability of a yeast artificial chromosome (YAC) vector, we decided to use this alternative source of large DNA fragments to obtain linkage. The technique involves the ligation of large (50-1,000 kilobase) genomic fragments into a vector that provides centromeric, telomeric and selective functions; the constructs are then introduced into Saccharomyces cerevisiae, and replicate in the same manner as the host chromosomes.     

Construction of a BAC library from cucumber （Cucumis sativus L.） and identification of linkage group specific clones

A bacterial artificial chromosome （BAC） library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber （Cucumis sativus L.） inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions （SCAR） and seven simple sequence repeats （SSR） markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction （PCR）. Fifteen markers gave at least two positive clones. As a result, 22 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.     

Comparison of human genetic and sequence-based physical maps

Recombination is the exchange of information between two homologous chromosomes during meiosis. The rate of recombination per nucleotide, which profoundly affects the evolution of chromosomal segments, is calculated by comparing genetic and physical maps. Human physical maps have been constructed using cytogenetics, overlapping DNA clones and radiation hybrids; but the ultimate and by far the most accurate physical map is the actual nucleotide sequence. The completion of the draft human genomic sequence provides us with the best opportunity yet to compare the genetic and physical maps. Here we describe our estimates of female, male and sex-average recombination rates for about 60% of the genome. Recombination rates varied greatly along each chromosome, from 0 to at least 9 centiMorgans per megabase (cM Mb(-1)). Among several sequence and marker parameters tested, only relative marker position along the metacentric chromosomes in males correlated strongly with recombination rate. We identified several chromosomal regions up to 6 Mb in length with particularly low (deserts) or high (jungles) recombination rates. Linkage disequilibrium was much more common and extended for greater distances in the deserts than in the jungles.     

Molecular cloning of human telomeres in yeast

Telomeres are the DNA sequences found at the ends of linear chromosomes. They define the boundaries of the genetical and physical maps of such chromosomes and so are particularly important for the complete mapping of large genomes that is now being attempted. Telomeres have been intensively studied in the yeast Saccharomyces cerevisiae and in ciliated protozoa: in these organisms the telomeric DNA consists of arrays of tandemly repeated short sequences in which one strand is guanosine-rich and oriented 5' to 3' towards the chromosome end. The conservation of these structural features is reflected in the observation that telomeric DNA from a variety of protozoa will function as telomeres on artificial linear mini-chromosomes in yeast. Tandem arrays of the sequence TTAGGG have been identified at the telomeres of humans and other mammals and also of trypanosomes. This indicates that the structural features of telomeres are conserved between higher and lower eukaryotes and implies that human telomeric DNA could function in yeast. I have used this idea to develop a strategy to isolate a specific human telomere as a molecular clone in yeast and have devised a simple and effective way of cloning other human telomeres and their associated sequences.     

Construction of a BAC library from cucumber (Cucumis sativus L.) and identification of linkage group specific clones

A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94; derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen markers gave at least two positive clones. As a result, 32 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.     

Construction of a BAC library from cucumber (Cucumis sativus L.) and identification of linkage group specific clones

A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94; derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen markers gave at least two positive clones. As a result, 32 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.     

Localisation of the G gamma-, A gamma-, delta- and beta-globin genes on the short arm of human chromosome 11.

Human-mouse somatic cell hybrids have proved invaluable in assigning human genes to their respective human chromosomes. To date, the success of this approach has depended on identifying human proteins which are synthesised in hybrid cells containing a small number of human chromosomes. Consequently, chromosome assignment has been limited mainly to human proteins which are expressed in man-mouse somatic cell hybrids and for which a suitable assay, usually electrophoretic or immunological, exists to distinguish between the human and murine homologous proteins. This technique is therefore unsuitable for the assignment of those human genes which are expressed only in differential cells and not in hybrid cells. Here, we describe how nucleic acid hybridisation and restriction endonuclease mapping of DNA can be combined to test for the presence of human structural gene sequences within hybrid cell DNA. This method can be used to assign any purified human DNA sequence to a human chromosome, and does not require the DNA sequence to be expressed in man-mouse hybrid cells.     

库拉索芦荟6号染色体表达序列标签的研究

将微分离的库拉索芦荟(Alove vera[L.] Burm.f)6号染色体DNA及库拉索芦荟叶总cDNA分别用LA-PCR(linker adaptor polymerase chain reaction)方法扩增,然后采用杂交片段扩增技术(hybrid specific amplification,HSA)分离6号染色体与cDNA同源的表达序列片段.对表达序列片段进行克隆,得到约40000个重组子.随机选取96个克隆进行分析,分别用DIG标记6号染色体的LA-PCR产物和cDNA扩增产物作探针对其进行点杂交,对显示阳性信号的克隆进行测序.将库拉索芦荟6号染色体的表达序列标签(expressed sequence tags, EST)与GenBank中核苷酸进行比较,发现库拉索芦荟6号染色体的表达序列中有87.4%与植物的EST具有同源性,12.6%的EST与人或动物的EST具有较弱同源性,其中与石刁柏的EST及非生物素胁迫下的小麦(Triticum aestivum)、咖啡(coffee)、高梁(Sorghum bicolor)和菠萝(pineapple)等作物的EST具有很高的同源性(＞93%).     

Development of specific chromosomal DNA pool for rice field eel and their application to gene mapping

The chromosomes 1, 3, 5, 6, 7, 10 and 12 of rice field eel (Monopterus albus Zuiew) have been microdissected successfully from meiosis Ⅰ diakinesis spreads by using glass microneedle under an inverted microscope. And the DOP-PCR products of the single chromosome dotted on the nylon membrane as “specific chromosomal DNA pool”, have been hybridized with 6 probes to map these genes. The mapping results show that Zfa has been mapped to chromosome 1, rDNA to chromosomes 3 and 7, both Gh and Pdegg to chromosome 10, Hsl to chromosome 5 and Hox genes have been detected on chromosomes 1, 3, 6 and 10 meantime. It has initiatively been suggested that chromosome 10 of rice field eel might possess the commom conserved synteny to that on chromosome 17 of human, chromosome 11 of mouse, chromosome 12 of pig and chromosome 19 of bovine. And so chromosome 3 of rice field eel might also contain the commom conserved synteny to that on chromosome 2 of zebrafish. Our study is an attempt to establish a new and feasible method to advance the study of gene mapping and chromosome evolution in fish, and also to provide a new idea to distinguish each chromosome on the base of molecular markers for fish.     

Construction of single-chromosome DNA library fromLilium regale Wilson

A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA fromLilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0.5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300–2500 base pairs (bp) with predominant fragments at about 1000 bp. Southern blot analysis confirmed that the PCR products originated from the genome ofLilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1800 bp, with an average of780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).     

DNA fingerprinting in birds

Several regions of the human genome are highly variable in populations because the number of repeats in these regions of a short 'minisatellite' sequence varies at high frequency. Different minisatellites have a core sequence in common, however, and probes made up of tandem repeats of this core sequence detect many highly variable DNA fragments in several species including humans, cats, dogs and mice. The hypervariable sequences detected in this way are dispersed in the genome and their variability means that they can be used as a DNA 'fingerprint', providing a novel method for the identification of individuals, confirmation of biological relationships and human genetic analysis. We show here that human minisatellite-derived probes also detect highly variable regions in bird DNAs. Segregation analysis in a house sparrow family confirms that these regions comprise many mostly heterozygous dispersed loci and we conclude that house sparrow DNA fingerprints are analogous to those of humans. Fingerprint analysis identified one nestling, with fingerprint bands not present in the parent pair's fingerprints, which we conclude resulted from an extrapair copulation. Extrabond copulations have been described in many wild bird species, but their success and hence adaptive significance have rarely been quantifiable. DNA fingerprinting will be of great significance to studies of the sociobiology, demography and ecology of wild birds.     

Cloning of the essential myotonic dystrophy region and mapping of the putative defect.

Myotonic dystrophy is a common dominant disorder (global incidence of 1:8,000) with variable onset and a protean nature of symptoms mainly involving progressive muscle wasting, myotonia and cataracts. To define the molecular defect, we have cloned the essential region of chromosome 19q13.3, including proximal and distal markers in a 700-kilobase contig formed by overlapping cosmids and yeast artificial chromosomes (YACs). The central part of the contig bridges an area of about 350 kilobases between two new flanking crossover borders. This segment has been extensively characterized through the isolation of five YAC clones and the subsequent subcloning in cosmids from which a detailed EcoRI, HindIII, MluI and NotI restriction map has been derived. Two genomic probes and two homologous complementary DNA probes were isolated using the cosmids. These probes are all situated within approximately 10 kilobases of genomic DNA and detect an unstable genomic segment in myotonic dystrophy patients. The length variation in this segment shows similarities to the instability seen at the fragile X locus. The physical map location and the genetic characteristics of the length polymorphism is compatible with a direct role in the pathogenesis of myotonic dystrophy.     

Isolation of a cDNA clone corresponding to an X-linked gene family (XLR) closely linked to the murine immunodeficiency disorder xid

The striking number of human and murine immunodeficiency disorders which map to the X chromosome suggests that genes localized on this chromosome must have important roles in lymphocyte development. At least seven distinct disorders in the human and two in the mouse disrupt lymphocyte maturation, particularly that of B cells, at characteristic stages. As functional genes mapping to the X chromosome in one mammal are found on the X chromosome in all other mammals, the same genes regulating lymphocyte development are expected to be found on the X chromosome in mouse and man. Investigations into the possible mechanisms of these X-linked disorders have been hampered by the lack of molecular probes for the genes or gene products affected; because of this, and the possibility of correlating one or more of the several hundred B- or T-cell-specific genes with a specific mutation, we surveyed 15 different B- and T-cell-specific cDNA clones for localization to the X chromosome. We report here the characterization of one of these murine cDNA clones, which hybridizes with a large, X-linked gene family, designated XLR (X-linked, lymphocyte-regulated). We show that the XLR gene family is closely linked to the X-linked immunodeficiency described in the CBA/N mouse strain (xid), by restriction fragment length polymorphism (RFLP) analysis of DNA from mice congeneic for xid. This finding, together with data on the expression of the XLR locus in B cells, indicates that this gene family either includes the locus defined by the xid mutation or is adjacent to it in a gene complex which may be important in lymphocyte differentiation.     

Construction and use of human chromosome jumping libraries from NotI-digested DNA

A basic difficulty in the molecular analysis of genes identified by mutations in the mammalian genome is the need to cover genetic distances corresponding to several hundred kilobases or more by molecular techniques like chromosome walking. In chromosome jumping, this limitation is overcome by the deletion of all but the extreme ends of large DNA molecules before cloning. We describe here the construction and characterization of a NotI 'jumping library' from human DNA. To characterize this library, random clones were analysed by restriction mapping. Clones carrying unique end fragments were characterized further by hybridization to Southern blots of NotI-cleaved human DNA separated on pulsed field gradient (PFG) gels. As a first step in a directional walk, the library was screened with a clone containing a NotI site cleaved in genomic DNA ('NotI linking clone') localized to the distal third of the short arm of human chromosome 4 (A.-M.F. & T.P., unpublished data). Starting and end points of two identified clones were positioned within a restriction map covering 850 kilobases.     

Construction and characterization of a bacterial artificial chromosome library of peach

This report briefly describes the construction and characterization of a peach [ prunus persica (L.) Batch] Var. Jingyu bacterial artificial chromosome (BAC) library. The variety Jingyu has many important agronomic characters of stone fruits, and it is a main parent in Chinese peach breeding. After cloning of the high molecular weight peach DNA into pBeloBAC 11, we obtained over 22 000 recombinant clones. The BAC library has an average insert size of 95 kb and represents approximately 7 times peach haploid genome equivalents. After being screened with two randomly amplified polymorphic DNA markers, W4 and P20, which are linked to yellow flesh and nectarine genes of peach respectively, ten positive clones have been detected. This library is very useful for map-based cloning of peach genes and physical mapping of peach genome.     

Closely related sequences on human X and Y chromosomes outside the pairing region

During meiosis the human X and Y chromosomes form a synaptonemal complex which covers most of Yp and the terminal 30% of Xp (ref. 1). By analogy with the autosomes, this is presumed to reflect DNA sequence homology. It has been suggested that these regions of the X and Y chromosomes contain either related or identical loci which are distal to a site of cross-over, and support for these ideas has come from the finding that an X-linked cell-surface antigen controlling gene MIC2 is related to a gene on the Y chromosome. A number of DNA sequences have been shown to occur either on the X and Y chromosomes or on the X, Y and autosomes. We have now isolated a sequence from the Y chromosome which is present on Xq and Yq. This region lies well outside the pairing segments, and sequence analysis reveals no base change in 1 kilobase pair (kb). This high degree of similarity between the X and Y chromosomes near the tips of the long arms is a strong indication that interchange can occur in this region.