cDNA surveying of specific tissue expression of human chromosome 19 sequences.

cDNA surveying is a straightforward approach for identifying sequences in genomic clones expressed in specific tissues. It has been applied to a subchromosomal region of human chromosome 19 (19q13.2-q13.4), a region that contains several known expressed sequences including the locus for myotonic dystrophy (DM). Genomic clones were selected from this region by probing a human placental cosmid library with a chromosome 19q-specific minisatellite sequence, or human genomic clones were isolated from a cosmid library constructed from a human chromosome 19q13.2-q13.3 hamster hybrid cell line using human repetitive DNA as probe. Pooled cDNAs synthesized from RNA of specific tissues characteristically affected in DM were depleted in repetitive sequences and used as hybridization probes against gridded cosmid arrays. DNA from the cDNA-positive cosmid clones was transferred to nylon filters and reprobed with cDNAs to identify restriction fragments that were expressed in these tissues. Hybridizing restriction fragments were subcloned, sequenced, and demonstrated to be nonrepetitive. Primer pairs complementary to subcloned sequences were constructed and used for PCR amplification of cDNA synthesized from RNA of tissues affected in myotonic dystrophy. PCR products were sequenced to verify the identity of expressed genomic DNA and its corresponding cDNA.     

A cosmid library specific for human Chromosome regions 6p21.3 and 6q27

We have explored the potential of irradiation-fusion gene transfer (IFGT) hybrids as a source of well-defined human chromosome fragments from which probes can be derived. Extensive characterization of the IFGT hybrid 4J4 with a full panel of markers from Chromosome (Chr) 6 showed that the human DNA content derives largely from 6p21.3 and 6q27. A cosmid library has been constructed from 4J4 DNA, and 370 recombinants containing human DNA have been isolated and overlapping clones ordered into 20 contigs. Regional localization of representative clones from each contig, determined by fluorescent in situ hybridization (FISH), places 13 contigs in 6q27 and 6 in 6p21.3. Preliminary screening of cDNA libraries with selected cosmids has identified two expressed sequences. Since there are a number of medically important genes in both these regions of human Chr 6 with several disease loci linked to the HLA-A region in 6p21.3 and various tumor suppressor genes to 6q27, this library will provide a valuable resource to aid the isolation of candidate genes for these diseases. In addition, unique markers for detailed physical and genetic mapping of these regions of human Chr 6 can be easily obtained.     

Studies on locus expansion, library representation, and chromosome walking using an efficient method to screen cosmid libraries

We have developed an efficient screening method to search for clones in cosmid libraries prepared from human genomic DNA. Genomic, cDNA, and cosmid probes have been used to isolate homologous cosmids from human chromosomes 7, 10, 16, 17 and X as part of a search for polymorphic nucleotide sequences. This method has been successfully applied to chromosome walking experiments at the interstitial retinol-binding protein locus on chromosome 10, and may be a useful tool for investigating representation of cloned sequences in cosmid libraries. Our library was prepared in the vector c2RB (Bates and Swift, 1983), but the method is applicable to any cosmid cloning system in which the inserted DNA can be separated from the vector by restriction enzyme digestion. A cosmid library containing five human genome equivalents can be rapidly screened using three to four Southern hybridization filters. This results in substantial labor saving, particularly when screening genomes of high complexity with many different probes. Another advantage of the system is that it allows for the long-term storage of the cosmids so that they can be screened whenever necessary. As a consequence, cosmid screening can be made a routine laboratory procedure.     

Assignment of 118 novel cDNAs of cynomolgus monkey brain to human chromosomes

In order to isolate genes that may not be represented in current human brain cDNA libraries, we have sequenced about 20,000 sequence tags of cDNA clones derived from cerebellum and parietal lobe of cynomolgus monkeys (Macaca fascicularis). We determined the entire cDNA sequence of approximately 700 clones whose 5'-terminal sequences showed no homology to annotated putative genes or expressed sequence tags in current databases of genetic information. From this, 118 clones with sequences encoding novel open reading frames of more than 100 amino acid residues were selected for further analysis. To localize the genes corresponding to these 118 newly identified cDNA clones on human chromosomes, we performed a homology search using the human genome sequence and fluorescent in situ hybridization. In total, 108 of 118 clones were successfully assigned to specific regions of human chromosomes. This result demonstrates that genes expressed in cynomolgus monkey are highly conserved throughout primate evolution, and that virtually all had human homologs. Furthermore, we will be able to discover novel human genes in the human genome using monkey homologs as probes.     

Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes

Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.     

Isolation of cDNA clones using yeast artificial chromosome probes.

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library.     

Isolation of Expressed Sequences Encoded by the Human Xq Terminal Portion Using Microclone Probes Generated by Laser Microdissection

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.     

人Xp11.2区4.3MbYAC重叠群：大尺度限制图与CpG岛分析

人Ｘｐ１１．２区域具有重要的医学遗传学和基础遗传学价值,它包含很多遗传疾病基因,且至少包含一个逃避Ｘ染色体失活的位点,非常规的基化多态也有发现。我们利用这一区域已知的一系列ＤＮＡ位标,从我们构建的ＹＡＣ库中筛选出一系列ＹＡＣ克隆。     

Clusters of genes encoding mouse transplantation antigens

We constructed a cosmid library from BALB/c mouse sperm DNA and isolated 64 cosmid clones with cDNA probes for transplantation antigens (class I molecules). Of these clones, 54 mapped into 13 gene clusters containing 36 distinct class I genes and encompassing 837 kilobases of DNA. One gene cluster mapped to the L region and a second cluster with seven genes to the Qa-2,3 region of the major histocompatibility complex. Restriction map and Southern blot analyses suggest that there are subgroups of class I genes. Using a 5' flanking sequence of the L gene as a hybridization probe, we show the L gene to be present in mouse strains expressing this antigen but deleted or mutated in strains failing to express it. Our data suggest that gene duplication and deletion presumably by homologous but unequal crossing-over has altered the size and organization of the class I clusters in different mouse strains and probably is an important mechanism for generating polymorphism in these genes. Analysis of the 36 class I genes with cDNA probes specific for the 5' and 3' ends shows that the exon encoding the third external domain is far more conserved than those encoding the first and second external domains of the transplantation antigen. These differences in variability have interesting functional implications.     

Searching for coding sequences in the mammalian genome: the H-2K region of the mouse MHC is replete with genes expressed in embryos.

We have searched for expressed genes in 170 kb of cosmid cloned DNA from the H-2K region of the mouse MHC. This region is known to contain two genes, H-2K and K2. We identified unique/low copy sequences evenly spaced along the cloned DNA, and used these as probes to search for conserved sequences in Southern blots from a variety of mammalian species. The majority of the unique sequences were found to have homologues and most of these were associated with CpG non-methylated islands. Northern blot analysis and isolation of clones from 5.5 and 10.5-day embryo cDNA libraries showed five additional genes encoded in the H-2K region. Four of these are abundant in embryos; the fifth is exclusively expressed in lymphoid cells. Our data indicate a minimum of seven genes in 170 kb, an unexpectedly high gene density. These results differ from two recent studies where similar lengths of cloned DNA were examined for expressed genes, and only one, or a part of one gene was found. The combined data suggest that the spatial organization of genes in the mammalian genome may not be random.     

Localization of 616 human chromosome 3-specific cosmids using a somatic cell hybrid deletion mapping panel.

A total of 5700 human chromosome 3-specific cosmid clones was isolated from a series of cosmid libraries constructed from somatic cell hybrids whose only human component was an entire chromosome 3 or a chromosome 3 containing an interstitial deletion removing 50% of long arm sequences. Several unique sequence chromosome 3-specific hybridization probes were isolated from each of 616 of these cosmids. These probes were then used to localize the cosmids by hybridization to a somatic cell hybrid deletion mapping panel capable of resolving chromosome 3 into nine distinct subregions. All 616 of the cosmids were localized to either the long or short arm of chromosome 3 and 63% of the short arm cosmids were more precisely localized. We have identified a total of 87 cosmids that contain fragments that are evolutionarily conserved. Fragments from these cosmids should prove useful in the identification of new chromosome 3-specific genes as well as in comparative mapping studies. The localized cosmids should provide excellent saturation of human chromosome 3 and facilitate the construction of physical and genetic linkage maps to identify various disease loci including Von Hippel Lindau disease and renal and small cell lung carcinoma.     

Isolation of a zinc finger motif (ZNF75) mapping on chromosome Xq26.

We report here the partial characterization of a new human zinc finger (ZNF75) gene of the Kruppel type mapping to the long arm of the X chromosome. A cosmid clone was isolated from a library specific to the Xq24-qter region by hybridization to a degenerate oligonucleotide representing the link between two contigous fingers of the C2H2 type. The sequence of the pertinent cosmid fragments demonstrated five consecutive zinc finger motifs, all pertaining to the Kruppel family. A reading frame starting at least 75 amino acids before the first zinc finger and ending 11 amino acids after the last one was identified; comparison with other ZF genes suggests that this genomic fragment represents the carboxy-terminal exon of the gene. Homology of approximately 55% in the zinc finger region was detected with many zinc finger genes including mouse Zfp-35 and human ZFN7 cDNA clones. Mapping using a panel of sematic cell hybrids and chromosomal in situ hybridization localized the gene to Xq26, in a region not previously known to contain zinc finger genes.     

Analysis of fibrinogen genes in patients with congenital afibrinogenemia

Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients. The results indicate that there is a single copy of the fibrinogen genes which are present and grossly intact in afibrinogenemic DNA.     

The human T cell receptor beta-chain gene complex contains at least 57 variable gene segments. Identification of six V beta genes in four new gene families.

The human TCR beta-chain gene complex includes at least 57 variable (V) gene segments, a number estimated using a combination of Southern blots of conventional and pulsed field gels, sequence analysis of cDNA clones, and from the analysis of genomic cosmid and phage clones. This number includes six TCR beta-chain V genes in four new families identified here by sequence analysis of clones derived from a human TCR beta-chain specific cDNA library. Comparison of the sequences of the new V beta genes with previously reported V beta sequences reveals predicted similarities but less than 75% nucleic acid identity that establishes them as new V beta families. One of the new V beta gene families includes three genes and the other three are single member families. Identification of these six new V beta genes falling into four V beta families brings the total number of transcribed human V beta families to 24 and makes it possible to refine the estimate of the total number of human TCR V beta genes to 57.     

Actin-like sequences are present on human X and Y chromosomes.

The human genome contains greater than 20 actin-related sequences, six of which at least are expressed as protein. We have shown by blot hybridization the presence of actin-like sequences on both the X and the Y chromosomes. These sequences can be detected in HindIII digests of genomic DNA, using as probe cDNA clones corresponding to human alpha skeletal actin or to a hamster (beta or gamma) cytoskeletal actin; they show more homology to the latter probe. The actin probes also detect a polymorphic DNA fragment showing autosomal inheritance with a frequency for the major allele of 0.55 in the population studied. The X-linked actin sequence has been assigned to a centromeric region between Xp11 and Xq11 by hybridization to DNAs from a panel of human-mouse hybrid cell lines, and thus lies outside the postulated region of homology between the X and Y chromosomes. The Y-linked actin sequence can serve as a marker to analyse anomalies of sex determination or of gametogenesis in man. It was found in all XY males studied but was absent from the genomic DNA of four unrelated 'XX male' subjects and two XX hermaphrodites. This shows that the region of chromosome Y which contains the actin sequence is not translocated onto the X chromosome (or onto autosomes) in these patients.     

Cloning of cDNA Derived from mRNA of the Electric Lobe of Torpedo marmorata and Selection of Putative Cholinergic-Specific Sequences

With the aim of selecting cDNA sequences expressed in neurons utilizing exclusively cholinergic synaptic transmission, cDNA derived from Torpedo electric lobe was cloned and screened by hybridization with probes originating from another brain region known for its low content of cholinergic neurons (cerebellum) and a nonneuronal tissue (muscle). This method led to the isolation of 18 clones among 3,200 showing no hybridization with probes other than those derived from electric lobe. These clones can therefore be considered to represent sequences involved in the expression of cholinergic function in neurons.