Major rearrangements in the alpha 5(IV) collagen gene in three patients with Alport syndrome.

The gene coding for the alpha 5 chian of type IV collagen (alpha 5(IV) collagen), which maps to Xq22, is a candidate gene for the X-linked dominant disease Alport syndrome (AS). Using three cDNA clones, covering the 3' end of the alpha 5(IV) collagen gene, 3 of 38 patients have been identified with mutations in this gene. Each of these patients shows a gross rearrangement of DNA: a deletion of at least 35 kb, an insertion/deletion event involving approximately 25 kb, and a duplication of at least 35 kb of DNA.     

Construction of a yeast artificial chromosome contig encompassing the human alpha 5(IV) collagen gene (COL4A5).

A PCR-based screening approach was used to isolate six yeast artificial chromosome (YAC) clones containing segments of the human alpha 5(IV) collagen gene (COL4A5). This gene is located at Xq22 and is known to be involved in the kidney disorder known as Alport syndrome (AS). By analyzing sequence-tagged sites, cDNA content, and rare-cutting restriction site patterns in these YAC clones, a contig that spans the entirety of the alpha 5(IV) gene was constructed. This contig may contain as much as 690 kb of DNA from the alpha 5(IV) locus. On the basis of the information obtained from these YAC clones, the genomic map and gene structure of the alpha 5(IV) gene have been refined. This study has also provided a valuable resource for subsequent studies of the alpha 5(IV) gene and its flanking DNA sequences.     

Isolation of mutations with dumpy-like phenotypes and of collagen genes in the nematode Pristionchus pacificus

The nematode Pristionchus pacificus was developed as a satellite system in evolutionary developmental biology and forward and reverse genetic approaches allow a detailed comparison of various developmental processes between P. pacificus and Caenorhabditis elegans. To facilitate map-based cloning in P. pacificus, a genome map was generated including a genetic linkage map of approximately 300 molecular markers and a physical map of 10,000 BAC clones. Here, we describe the isolation and characterization of more than 40 morphological mutations that can be used as genetic markers. These mutations fall into 12 Dumpy genes and one Roller gene that represent morphological markers for all six P. pacificus chromosomes. Using an in silico approach, we identified approximately 150 hits of P. pacificus collagen genes in the available EST, BAC-end, and fosmid-end sequences. However, 1:1 orthologs could only be identified for fewer than 20 collagen genes.     

Cloning and chromosomal localization of human genes encoding the three chains of type VI collagen.

Type VI collagen is a heterotrimer composed of three polypeptide chains, alpha 1(VI), alpha 2(VI), and alpha 3(VI). By immunological screening of an expression cDNA library, human cDNAs specific for each chain were isolated and characterized. Major mRNA species encoding these chains have a size of 4.2 kb (alpha 1), 3.5 kb (alpha 2), and 8.5 kb (alpha 3). The cDNA clones were also used to map the genes on human chromosomes by somatic cell hybrid analysis and in situ hybridization. The alpha 1 (VI) and alpha 2(VI) collagen genes were both located on chromosome 21, in band q223. This represents a third example of a possible physical proximity of two collagen loci. The alpha 3(VI) collagen gene was localized to chromosome 2, in the region 2q37. The alpha 3(VI) collagen gene is the fifth extracellular matrix gene to be localized to 2q, as four other extracellular matrix genes--i.e., the alpha 1(III) and alpha 2(V) collagen genes, the elastin gene, and the fibronectin gene--have been previously mapped to the distal region of the long arm of chromosome 2.     

Linkage mapping and partial sequencing of 10 cDNA loci in the chicken

Ten cDNA clones derived from chicken spleen cell mRNA have been partially sequenced and the genes which encode the mRNAs have been located within the linkage map of the chicken genome. The sequences of five of these clones show strong homology to known mammalian genes, the remainder show little homology to sequences present in the current databases. Interestingly, one of these clones appears to be the chicken homologue of the mammalian peptide transporter gene TAP2 and is located within the major histocompatibility complex. Two other clones are homologous to genes involved in protein synthesis and these are tightly linked in chickens, as in mice. These results suggest that partial sequencing and mapping of clones from selective cDNA libraries may be an efficient way of adding candidate genes to the chicken linkage map and that on a local scale there may be some conservation of grouping of genes between chickens and mammalian species.     

A first generation physical map of the medaka genome in BACs essential for positional cloning and clone-by-clone based genomic sequencing

In order to realize the full potential of the medaka as a model system for developmental biology and genetics, characterized genomic resources need to be established, culminating in the sequence of the medaka genome. To facilitate the map-based cloning of genes underlying induced mutations and to provide templates for clone-based genomic sequencing, we have created a first-generation physical map of the medaka genome in bacterial artificial chromosome (BAC) clones. In particular, we exploited the synteny to the closely related genome of the pufferfish, Takifugu rubripes, by marker content mapping. As a first step, we clustered 103,144 public medaka EST sequences to obtain a set of 21,121 non-redundant sequence entities. Avoiding oversampling of gene-dense regions, 11,254 of EST clusters were successfully matched against the draft sequence of the fugu genome, and 2363 genes were selected for the BAC map project. We designed 35mer oligonucleotide probes from the selected genes and hybridized them against 64,500 BAC clones of strains Cab and Hd-rR, representing 14-fold coverage of the medaka genome. Our data set is further supplemented with 437 results generated from PCR-amplified inserts of medaka cDNA clones and BAC end-fragment markers. Our current, edited, first generation medaka BAC map consists of 902 map segments that cover about 74% of the medaka genome. The map contains 2721 markers. Of these, 2534 are from expressed sequences, equivalent to a non-redundant set of 2328 loci. The 934 markers (724 different) are anchored to the medaka genetic map. Thus, genetic map assignments provide immediate access to underlying clones and contigs, simplifying molecular access to candidate gene regions and their characterization.     

Cloning and sequencing of pro-alpha 1 (XI) collagen cDNA demonstrates that type XI belongs to the fibrillar class of collagens and reveals that the expression of the gene is not restricted to cartilagenous tissue

We have isolated several overlapping cDNA clones encoding alpha 1(XI) collagen chains from human and rat cDNA libraries. Together the human cDNAs code for 335 uninterrupted Gly-X-Y triplets, and a 264-amino acid C-propeptide, while the rat cDNAs cover the entire C-propeptide and about a third of the triple-helical domain. Comparison of the human and rodent nucleotide sequences showed a 95% sequence similarity. The identification of the clones as alpha 1(XI) cDNAs was based on the complete identity between the amino acid sequences of three human alpha 1(XI) cyanogen bromide peptides and the cDNA-derived sequence. Examination of and the cDNA-derived amino acid sequence showed a variety of structural features characteristic of fibrillar-forming collagens. In addition, nucleotide sequence analysis of a selected portion of the corresponding human gene revealed the characteristic 54-base pair exon motif. We conclude therefore that pro-alpha 1 (XI) collagen belongs to the group of fibrillar collagen genes. We also suggest that the expression of this gene is not restricted to cartilage, as previously thought, since the cDNA libraries from which the clones were isolated, originated from both cartilagenous and noncartilaginous tissues.     

Molecular cloning of alpha 5(IV) collagen and assignment of the gene to the region of the X chromosome containing the Alport syndrome locus.

Type IV collagen is a major structural component of basement membranes. Four constituent polypeptides have been described and characterized to different degrees. Whereas the primary structure of the alpha 1(IV) and alpha 2(IV) chains has been completely established, only short protein sequences have been reported for the recently recognized alpha 3(IV) and alpha 4(IV) subunits. We have isolated overlapping human cDNA clones whose derived amino acid sequence is highly homologous to the alpha 1(IV) and alpha 2(IV) chains. However, these clones code for neither alpha 3(IV) nor alpha 4(IV), and thus this new polypeptide has been designated the alpha 5 chain of type IV collagen. To determine whether the gene encoding the alpha 5(IV) chain is syntenic with the contiguously arranged alpha 1(IV) and alpha 2(IV) genes at 13q34, the alpha 5(IV) cloned DNA was hybridized to genomic DNA from somatic cell hybrids and to metaphase chromosomes. The results demonstrated that the alpha 5(IV) collagen gene is located on the long arm of the X chromosome. Since 14 collagen genes have previously been assigned to nine autosomes, these data represent the first mapping of a collagen gene to the X chromosome. Most important, the alpha 5(IV) gene has been sublocalized to bands Xq22----q23, which are in the same region known to contain the locus for the X-linked form of Alport syndrome. It is therefore possible that this severe dominantly inherited nephritis, manifested by splitting of the glomerular basement membrane, could be caused by mutations in the alpha 5(IV) collagen gene.     

Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts.     

RFLP mapping of partially sequenced leaf cDNA clones in maize

We report here the results of mapping a set of 92 leaf cDNA clones in maize. The ends of each of these cDNA clones have previously been partially sequenced, and the sequence comparison has revealed the putative function for 28 clones. It is expected that the RFLP map developed using these expressed sequence tags will be of great importance for future maize genome analysis, such as for PCR-based gene mapping or gene function identification.Contribution from the Missouri Agricultural Experiment Station. Journal Series N. 12,019.     

Analysis by molecular cloning of the human class II genes

The HLA class II genes control immune responsiveness to defined antigens; they encode cell surface heterodimers composed of alpha and beta glycopeptides. Recently, cDNA and genomic clones encoding these chains have been isolated, which allows molecular analysis of the class II genes. cDNA clones encoding the alpha chain of the HLA-DR antigen as well as that of another HLA class II antigen have been identified and characterized by nucleotide sequence analysis. These clones have been used as probes to isolate additional class II alpha cDNA clones in cDNA libraries and to identify polymorphisms in genomic DNA. Polymorphic restriction sites have been localized within the HLA-DR alpha gene and used as genetic markers in the analysis of families and of disease (insulin-dependent diabetes mellitus) and control populations. In addition, cDNA clones encoding the DR beta and DC beta chains were used as hybridization probes to identify DNA polymorphism. cDNA clones encoding the DR gamma (Ii) chain have also been identified; unlike the DR alpha and DR beta loci, the DR gamma gene is located on some chromosome other than chromosome 6. The genetic complexity of the human class II alpha and beta loci, as revealed by analysis with cDNA and genomic clones, is greater than that of the murine class II genes. The extent of that complexity will be defined by future work in this area.     

Identification and characterization of three genes and two pseudogenes on chromosome 13

A study was conducted on the feasibility of isolating genes and pseudogenes that map to chromosome 13 by a hybridization-based approach using a 13-specific library and pools of repeat-free cDNA clones. Five pairs of cDNA and chromosome 13 genomic clones were identified and characterized. Partial or full-length sequence was derived from all cDNAs, and database searches were performed for putative gene identification. Partial sequence was also obtained from the chromosome 13 genomic clones for comparison with those of the hybridizing cDNAs. As a result of these analyses we identified three genes, a putative homologue of a porcine mRNA encoding an unidentified hepatic protein, a putative homologue of a yeast integral membrane protein, and a gene for a translationally controlled tumor protein, and two processed pseudogenes, ribosomal proteins L23a and S3a. The latter was formerly identified as the v-fos transformation effector gene, Fte-1, and recently cited as a possible candidate for the BRCA2 gene on chromosome 13. All genes and pseudogenes were localized to cytogenetic bands by in situ hybridization of metaphase chromosomes with probes derived from the chromosome 13 genomic clones.     

Large-scale EST sequencing in rice

Large-scale cDNA analysis provides several great advantages for genome investigations in rice. Isolated and partially characterized cDNA clones have contributed not only to the construction of an RFLP linkage map and physical maps of the chromosomes but also to investigations of the mechanisms of expression of various isozymes and family genes. The ultimate aim of our large-scale cDNA analysis is to catalogue all the expressed genes of this important cereal, including tissue-specific, developmental stage-specific, and stress-specific genes. As of August 1996, the Rice Genome Research Program (RGP) has isolated and partially sequenced more than 29000 cDNA clones from various tissues and calluses in rice (Nipponbare, a japonica variety). The sequence data were translated into amino acid sequences for the 3 possible reading frames, and the similarity of these amino acid sequences to known proteins registered in PIR were examined. About 25% of the clones had significant similarities to known proteins. Some of the hit clones showed library-specific distributions, indicating that the composition of the clones in each library reflects, to some extent, the regulation of gene expression specific to differentiation, growth condition, or environmental stress. To further characterize the cDNA clones, including unknown clones, nucleotide sequence similarities of 24728 clones were analyzed and the clones were classified into around 10000 independent groups, suggesting that around a half or one third of expressed genes in rice have already been captured. These results obtained from our large-scale cDNA analysis provide useful information related to gene expression and regulation in rice.