人体基因组随机单拷贝顺序的分离及其在染色体上的定位

本文从构建杂种细胞14-7-1的基因组文库出发,用种特异的探针分离出含有人体基因组顺序的重组子,并进一步分析了其中13个克隆,得到8个单拷贝顺序。通过与已建立的杂种细胞克隆分布板杂交以及染色体的原位杂交方法,将1个单拷贝顺序FD11-1定位在11p11-q11上。由于已经报道在11号染色体上具有3个连锁群,它们分别位于11p15、11p13和11q13上,因此,FD11-1有可能为11号染色体连锁基因图的建立提供1个有意义的座位。     

A comparative map of chicken chromosome 24 and human chromosome 11

To improve the physical and comparative map of chicken chromosome 24 (GGA24; former linkage group E49C20W21) bacterial artificial chromosome (BAC) contigs were constructed around loci previously mapped on this chromosome by linkage analysis. The BAC clones were used for both sample sequencing and BAC end sequencing. Sequence tagged site (STS) markers derived from the BAC end sequences were used for chromosome walking. In total 191 BAC clones were isolated, covering almost 30% of GGA24, and 76 STS were developed (65 STS derived from BAC end sequences and 11 STS derived within genes). The partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases, and revealed matches to 19 genes, expressed sequence tags (ESTs) and genomic clones located on human chromosome 11q22-q24 and mouse chromosome 9. Furthermore, 11 chicken orthologues of human genes located on HSA11q22-q24 were directly mapped within BAC contigs of GGA24. These results provide a better alignment of GGA24 with the corresponding regions in human and mouse and identify several intrachromosomal rearrangements between chicken and mammals.     

Fine mapping of the muscular dystrophy (AM) gene on chicken chromosome 2q

Our previous studies revealed that the genetic locus for chicken muscular dystrophy of abnormal muscle (AM) mapped to chromosome 2q, and that the region showed conserved synteny with human chromosome 8q11-24.3. In the current study, we mapped the chicken orthologues of genes from human chromosome 8q11-24 in order to identify the responsible gene. Polymorphisms in the chicken orthologues were identified in the parents of the resource family. Twenty-three genes and expressed sequence tags (ESTs) were mapped to chicken chromosome 2 by linkage analysis. The detailed comparative map shows a high conservation of synteny between chicken chromosome 2q and human chromosome 8q. The AM locus was mapped between [inositol(myo)-1(or4)-monophosphatase 1] (IMPA1) gene and [core-binding factor, runt domain, alpha-subunit 2; translocated to 1; cyclin D-related] (CBFA2T1) gene. The genes located between IMPA1 and CBFA2T1 are the most likely candidates for chicken muscular dystrophy.     

The mouse alpha 1(XII) and human alpha 1(XII)-like collagen genes are localized on mouse chromosome 9 and human chromosome 6.

Type XII collagen is a member of the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix proteins. Like the other members of this group, collagen types IX and XIV, type XII has alternating triple-helical and non-triple-helical domains. Because of its structure, its association with collagen fibrils, and its distribution in dense connective tissues, type XII is thought possibly to act as a cross-bridge between fibrils and resist shear forces caused by tension. A portion of the ffuse gene was isolated by screening a genomic library with a chicken alpha 1 (XII) cDNA probe, followed by subcloning and sequence analysis. Comparison of exon sequences with the sequence of a mouse cDNA clone allowed the mouse gene to be identified as the alpha 1 (XII) collagen gene. In the mouse, Col12a1 is located on chromosome 9, as determined by linkage analysis using DNA from interspecific backcrosses with Mus spretus. Screening of a human genomic library also allowed the isolation of a human alpha 1(XII)-like gene (CoL12A1). This gene was mapped to chromosome 6 by blot hybridization to DNA from human/hamster hybrid cell lines. This information should prove useful in determining the role of type XII collagen genes as candidate genes in inheritable connective tissue diseases.     

Localization of the Homolog of a Mouse Craniofacial Mutant to Human Chromosome 18q11 and Evaluation of Linkage to Human CLP and CPO

The transgene-induced mutation 9257 and the spontaneous mutation twirler cause craniofacial and inner ear malformations and are located on mouse chromosome 18 near the ataxia locusax.To map the human homolog of 9257, a probe from the transgene insertion site was used to screen a human genomic library. Analysis of a cross-hybridizing human clone identified a 3-kb conserved sequence block that does not appear to contain protein coding sequence. Analysis of somatic cell hybrid panels assigned the human locus to 18q11. The polymorphic microsatellite markers D18S1001 and D18S1002 were isolated from the human locus and mapped by linkage analysis using the CEPH pedigrees. The 9257 locus maps close to the centromeres of human chromosome 18q and mouse chromosome 18 at the proximal end of a conserved linkage group. To evaluate the role of this locus in human craniofacial disorders, linkage to D18S1002 was tested in 11 families with autosomal dominant nonsyndromic cleft lip and palate and 3 families with autosomal dominant cleft palate only. Obligatory recombinants were observed in 8 of the families, and negative lod scores from the other families indicated that these disorders are not linked to the chromosome 18 loci.     

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.     

Cloning, genomic sequence, and chromosome mapping of Scyb11, the murine homologue of SCYB11 (alias betaR1/H174/SCYB9B/I-TAC/IP-9/CXCL11)

A T-cell attracting CXC chemokine phylogenetically related to MIG and SCYB10 was recently characterized and termed SCYB11 (alias betaR1/H174/SCYB9B/I-TAC/IP-9/CXCL11). Here, we cloned the cDNA of the murine homologue of this protein, Scyb11, from interferon-gamma/lipopolysaccharide-stimulated RAW264.7 mouse macrophage-like cells. The nucleotide sequence of Scyb11 shares 63% identity with its human counterpart. It encodes a 100 amino acid immature protein of 11,265 Da which contains a putative signal peptide of 21 amino acids. The molecular mass of the mature protein was calculated to be 9,113 Da. Sequence identity of the murine and human SCYB11 proteins is 68%. Phylogenetic tree analysis of mouse CXC chemokines places SCYB11 together with the murine homologues of MIG and SCYB10 (Crg-2/muIP-10) on an individual branch. A genomic sequence was obtained by genome walking and subcloning DNA fragments from a BAC clone containing Scyb11. Like human SCYB11, Scyb11 contains 4 exons with intron/exon boundaries at positions comparable to the human gene. Whereas introns 2 and 3 are of similar length in the murine and human genes, intron 1 of Scyb11 contains 1,260 bp more than intron 1 of the human gene. Intron 1 of Scyb11 is also characterized by a 201-bp stretch with repetitive sequences of high cryptic simplicity. Using a BAC clone containing Scyb11, this gene could be mapped to chromosome 5 at position 5E3. Since human SCYB11 is localized on 4q21.2, this result confirms the mouse/human homology of the two chromosome regions.     

Isolation, mapping, and characterization of two cDNA clones expressed in the cerebellum.

In an effort to characterize genes expressed in the cerebellum, we have isolated two cDNA clones, H11B (D16S286) and 507 (D5S344), that hybridized to a cerebellar cDNA probe. Using a panel of human-rodent somatic cell hybrids, cDNA clone H11B was mapped to human chromosome 16, and clone 507 was mapped to human chromosome 5. TaqI RFLPs were identified with both clones and were used for linkage analysis in the CEPH families. D16S286 was tightly linked to several markers near chromosome 16p13, and D5S344 was tightly linked to several markers on chromosome 5q. Sequence tagged sites or expressed sequence tags were generated from the 3' untranslated regions of both cDNA clones.     

Molecular cloning and chromosomal mapping of CCND genes encoding human D-type cyclins.

A human D-type cyclin gene (CCND1/cyclin D1/PRAD1) was previously isolated by virtue of its ability to complement a triple G1 cyclin (Cln) deficiency of Saccharomyces cerevisiae and was also identified as a candidate BCL1 oncogene. We now report the molecular cloning of two additional human D-type cyclin genes, CCND2 (cyclin D2) and CCND3 (cyclin D3). All three human D-type cyclin genes encode small (33-34 kDa) proteins that share an average of 57% identity over the entire coding region and 78% in the cyclin box. The D-type cyclins are most closely related to cyclin A (39% identity) and cyclin E (36%), followed by cyclin B (29%) and cyclin C (21%). Isolation and characterization of genomic clones revealed two pseudogenes corresponding to CCND2 and CCND3, respectively. All three cyclin D genes are interrupted by an intron at the same position. CCND2 has been mapped to chromosome 12p13, and CCND3 has been mapped to chromosome 6p21.     

The zebrafish fth1, slc3a2, men1, pc, fgf3 and cycd1 genes define two regions of conserved synteny between linkage group 7 and human chromosome 11q13

In addition to being an excellent model system for studying vertebrate development, the zebrafish has become a great tool for gene discovery by mutational analysis. The recent availability of the zebrafish EST database and radiation hybrid mapping panels has dramatically expanded the framework for genomic research in this species. Developing comparative maps of the zebrafish and human genomes is of particular importance for zebrafish mutagenesis studies in which human orthologs are sought for zebrafish genes. However, only partial cDNA sequences are determined routinely for mapped ESTs, leaving the identity of the EST in question. It previously had been reported that zebrafish linkage group 7 shares conserved synteny with human chromosome 11q13. In an effort to further define this relationship, five full-length zebrafish cDNAs, fth1, slc3a2, prkri, cd81, and pc, as well as one putative human gene, DBX were identified and their map positions ascertained. These six genes, along with men1, fgf3 and cycd1 define two regions of conserved synteny between linkage group 7 and 11q13.     

Mapping of rabbit chromosome 1 markers generated from a microsatellite-enriched chromosome-specific library

A genomic DNA library was produced from flow-sorted rabbit chromosome 1 and enriched for fragments containing CA-repeats. Clones containing CA-repeats were identified and primers for amplification of the microsatellite were developed after sequencing the clone. The degree of polymorphism was tested in rabbits from different breeds. This approach identified 12 microsatellite markers which could be used for studying linkage relationships in the progeny of an F(2)-intercross: (AX/JUxIIIVO/JU) F(2), and two backcrosses: (OS/JxX/J)X/J and (WH/JxX/J)X/J. Seven of these markers were mapped on chromosome 1.     

cDNA cloning and mapping of the human creatine kinase M gene to 19q13

We describe the first isolation of a human creatine kinase M cDNA clone and its mapping of the gene to human chromosome 19. A human creatine kinase M cDNA clone, pJN2CK-M, harboring a 1,160-bp insert, was isolated by colony hybridization with a previously sequenced chicken creatine kinase M cDNA probe. The human cDNA was used as a probe in Southern transfers of TaqI-digested genomic DNA from mouse/human somatic-cell hybrids to localize the human creatine kinase-M gene to chromosome 19. In situ hybridization of the tritiated cDNA probe to metaphase chromosomes of peripheral blood lymphocytes from normal males revealed significant labeling to chromosome 19. These two independent methodologies assign the human creatine kinase-M gene to chromosome 19. Since greater than 69% of the grains of chromosome 19 label band q13, the human creatine kinase-M gene has been mapped to 19q13. On the basis of high-resolution G-banding, the predominant labeling site was 19q13.2-q13.3.     

Genomic organization and assignment of VAMP2 to 17p12 by FISH

We describe the complete sequence, genomic organization, and FISH chromosome mapping of the human VAMP2. We identified a 7-kb clone, pISSHG2b3A, containing the entire structure of VAMP2. Previous studies performed by others identified a 5-kb clone, pVPC5-2, containing the incomplete VAMP2. The pVPC5-2 clone was partially sequenced and mapped to the broad region 17pter-->p12 by somatic cell hybridization. Our clone overlaps the pVPC5-2 clone and extends approximately 2 kb at the 3' end. In this study, we mapped this gene more precisely on 17p12 by FISH and we found a new polymorphic microsatellite, (GT)(7)CC(GT)(5), in exon V. This microsatellite, revealing three alleles with frequencies of 0.778, 0.139, and 0.083, might be useful for future linkage studies. Finally, we localized three previously known markers, stSG12859, TIGR-A002F11, and WIAF-1699 (alias stSG4044), in the 3' untranslated region of the gene.     

Close linkage of genes encoding receptors for subgroups A and C of avian sarcoma/leucosis virus on chicken chromosome 28

Avian sarcoma and leucosis viruses (ASLV) are classified into six major subgroups (A to E and J) according to the properties of the viral envelope proteins and the usage of cellular receptors for virus entry. Subgroup A and B receptors are identified molecularly and their genomic positions TVA and TVB are mapped. The subgroup C receptor is unknown, its genomic locus TVC is reported to be genetically linked to TVA, which resides on chicken chromosome 28. In this study, we used two chicken inbred lines that carry different alleles coding for resistance (TVC(R) and sensitivity (TVC(S)) to infection by subgroup C viruses. A backross population of these lines was tested for susceptibility to subgroup C infection and genotyped for markers from chicken chromosome 28. We confirmed the close linkage between TVA and TVC loci. Further, we have described the position of TVC on chromosome 28 relative to markers from the consensus map of the chicken genome.     

Isolation and characterization of DNA sequences from flow-sorted human chromosome 22 libraries

Two flow-sorted chromosome 22 libraries were used to isolate DNA sequences specific for chromosome 22. 45-phage DNAs were probed against human genomic DNA. 12 of them showed unique or low-copy character. Using digested DNA from rodent-human hybrid cell lines, 3 of the 12 recombinants were assigned unique to chromosome 22 and regionally mapped. 1 clone mapped to 22pter-q11, 1 clone to 22q12-qter and 1 clone, for which in situ hybridization was performed, to 22q13.1. 2 low-copy probes, 1 of them displaying polymorphisms in MspI and TaqI digests of individual DNAs, must have similar sequences on 22 and additional chromosomes. Furthermore, a highly repetitive DNA representing a compound locus of some hundred kilobases on chromosome 22 was isolated. These 6 probes may provide useful tools for studying the structure and function of this small chromosome involved in a relatively high number of inherited and acquired diseases.