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The recessive aphakia (ak) mouse mutant is characterized by bilateral microphthalmia due to a failure of lens morphogenesis. We fine-mapped the ak locus to the interval between D19Umi1 and D19Mit9, developed new polymorphic markers, and mapped candidate genes by construction of a BAC contig. The Pitx3 gene, known to be expressed in lens primordia, shows zero recombination with the ak mutation on our intersubspecific intercross panel representing 1170 meioses. A recent report described a deletion in the intergenic region between Gbf1 and Pitx3 as the possible ak mutation. Our results differ in that we find not only the distant intergenic deletion, but also a much larger deletion directly in the Pitx3 gene, eliminating exon 1 and extending into intron 1 and the promoter region. Pitx3 transcript levels are severely reduced in ak/ak mice from E11.5 to newborn (5 +/- 1% of the wildtype levels at E13.5), while an involvement of the flanking Gbf1 and Cig30 genes in the aberrant lens development is highly unlikely based on expression analysis. We conclude that the ak mutation consists of two deletions, the larger of which removes part of Pitx3, indicating a crucial role of this gene in early lens development., (Copyright 2001 Academic Press.)

Vascular anomalies are congenital lesions that usually occur sporadically, but can be inherited. Previously, we have described that venous malformations, localized bluish-purple skin lesions, are caused by an activating mutation in the TIE2/TEK receptor. Moreover, we mapped another locus to chromosome 1p21-p22, for venous malformations with glomus cells (VM-GLOM). Here we report a physical map, based on 18 overlapping YAC clones, spanning this 5-Mb VMGLOM locus, from marker GATA63C06 to D1S2664. In addition, we report a sequence-ready PAC map of 46 clones covering 1.48 Mb within the YAC contig, a region to which we have restricted VMGLOM. We describe 21 new STSs and nine novel CA repeats, seven of which are polymorphic. These data will enable positional cloning of genes for diseases mapped to this locus, including the VMGLOM gene, likely a currently unknown regulator of vasculogenesis and/or angiogenesis.

Spt3 is a Saccharomyces cerevisiae transcription factor that is required in vivo for the transcription of a number of RNA polymerase II-transcribed genes. We report the cloning of the gene encoding the human homologue of Spt3, SUPT3H, and its initial functional analysis. The human and yeast Spt3 homologues share an overall identity of 30% that defines three conserved regions, suggesting possible functional domains. To determine whether SUPT3H is a true functional Spt3 homologue, we tested for complementation of an spt3Delta mutation in yeast. While expression of the full-length SUPT3H is unable to complement an spt3Delta mutation, expression of a human-yeast chimeric gene that contains 42% human sequences can partially complement an spt3Delta mutation. These data suggest that the transcriptional control carried out by Spt3 has been conserved from yeast to human., (Copyright 1998 Academic Press.)

Overlapping cDNA clones that encode the full-length human alpha 1(XII) collagen polypeptides were isolated. The long variant molecule cDNA of 9750 nucleotides (nt) contains a 9189-nt open reading frame encoding 3063 amino acid residues. The short variant molecule cDNA of 6258 nt contains a 5697-nt open reading frame encoding 1899 amino acid residues. At the amino terminus of each variant is a 24-residue signal peptide that is followed by the mature polypeptides of 3039 amino acid residues with a calculated molecular mass of 330,759 Da for the long variant and 1875 amino acid residues with a calculated molecular mass of 203,163 Da for the short variant polypeptide. The human collagen XII chains are predicted to have all the structural domains described for the molecules in chicken and mouse, including, fibronectin type III repeats, von Willebrand factor A domains, and two triple-helical domains similar to those of all the other collagen family members. The amino acid residue sequence of human alpha 1(XII) collagen showed 92% identity to the mouse chain and 78% identity to the chicken chain. The sequence of three peptide fragments of collagen XII isolated from human placenta was identical to the sequence predicted from the deduced cDNA sequence and confirms that the cDNA encodes human alpha 1(XII) collagen. An isolated genomic clone was used to map the locus of the COL12A1 gene to chromosome 6q12-q13, very close to the locus of the FACIT collagen genes COL9A1 and COL19A1. RT-PCR on a variety of cDNAs demonstrates that both variant transcripts appear in human amnion, chorion, skeletal muscle, small intestine, and in cell cultures of human dermal fibroblasts, keratinocytes, and endothelial cells. Only the small variant transcript is apparent in human lung, placenta, kidney, and a squamous cell carcinoma cell line. These results confirm the previous observations showing that collagen XII is found in collagen I-containing tissues.

The membrane-type matrix metalloproteinases (MT-MMPs) constitute a newly discovered family of four enzymes within the matrix metalloproteinase (MMP) superfamily. We have mapped the genes for MT1-MMP (MMP14), MT2-MMP (MMP15), and MT3-MMP (MMP16) using in situ hybridization to human metaphase chromosomes. In contrast to the genes for many MMPs that are clustered on chromosome 11, the genes MMP14, MMP15, and MMP16 all map to distinct chromosomes. MMP14 maps to human chromosome 14, MMP15 to human chromosome 16, and MMP16 to human chromosome 8.

Type IX collagen is composed of three polypeptides derived from the human genes COL9A1, COL9A2, and COL9A3 that assemble to form a mature collagen molecule with the structure alpha 1(IX)alpha 2(IX)alpha 3(IX). We have identified overlapping cDNA and genomic clones that encode for the entire alpha 3 chain of human type IX collagen. Tryptic peptides from the human alpha 3(IX) collagen chain were subjected to N-terminal amino acid sequencing, and a stretch of 124 contiguous amino acids that included the NC1, COL1, and NC2 domains was obtained. Degenerate oligonucleotide primers were designed based on the amino acid sequences of the human tryptic peptides as well as bovine peptides and sequences from chicken cDNA clones. These primers were used to amplify three overlapping PCR products that covered the majority of the human alpha 3(IX) collagen. PCR products were then used to identify overlapping cDNA clones from a human chondrocyte library. A lambda genomic clone was identified that contained the 5'-most exon that encodes the signal peptide to complete the entire structure of the human alpha 3(IX) collagen chain. Genomic amplification identified a single-strand conformational polymorphism in COL1 that was used to map COL9A3 to chromosome 20q13.3 by linkage analysis. The present study completes the structure of human type IX collagen, and linkage for COL9A3 completes the genomic mapping of cartilage collagen genes. These data will greatly assist the genetic screening of families with degenerative cartilage and eye diseases by allowing investigators to screen for a complete set of candidate collagen gene markers.

The BAX gene is a member of the Bcl-2 gene family; it encodes a 21-kDa protein whose association with Bcl-2 is believed to play a critical role in regulating apoptosis. Through analysis of human-hamster somatic cell hybrid DNA and by in situ hybridization to metaphase chromosomes, we have determined that the human BAX gene is located in the q13.3-q13.4 region of human chromosome 19. We have also isolated a BAX cDNA clone in which that part of the mRNA encoded by exon 3 is absent. The skipping of exon 3 and the resultant splicing of exons 2 and 4 maintains the original reading frame and predicts the existence of an interstitially truncated form of the major Bax protein (Bax alpha), termed Bax delta. Unlike two previously described variant forms of Bax alpha (Bax beta and Bax tau), Bax delta retains the functionally critical C-terminal membrane anchor region as well as the Bcl-2 homology 1 and 2 (BH1 and BH2) domains.

We have determined the chromosomal locations of the human and murine genes coding for alpha 2(IX) collagen, a polypeptide subunit of the heterotrimeric type IX collagen molecule. COL9A2 was mapped to human chromosome 1p32.3-p33 using fluorescence in situ hybridization. A single-strand conformational polymorphism within the murine Col9a2 gene was used to map this locus to mouse chromosome 4. We also present new sequence data, which completes the coding information for the human alpha 2(IX) chain and revises the sequence for the chicken alpha 2(IX) chain. This permits comparison of the carboxyl-terminal (NC1) domains of the alpha 1(IX), alpha 2(IX), and alpha 3(IX) chains across several species.

Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which we have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, we report the isolation of human cDNAs and genomic DNAs encoding the alpha 1(XVIII) collagen chain. Utilizing a genomic clone as probe, we have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, we have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2.

The tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of the matrix metalloproteinases, a group of zinc-binding endopeptidases involved in the degradation of the extracellular matrix. We have isolated overlapping cDNAs encoding a novel human TIMP, TIMP-3. The cDNAs contain a 591-bp-long open reading frame encoding 9 amino acid residues of the signal peptide and 188 residues of the mature TIMP-3 polypeptide. Both the nucleotide sequence and the conceptual translation product of the human TIMP3 cDNA have a high degree of similarity to ChIMP-3, a recently cloned metalloproteinase inhibitor in the chicken, and to the TIMP1 and TIMP2 gene products, including 12 conserved cysteinyl residues at the same relative positions. The TIMP3 gene is expressed in many tissues, with highest expression in the placenta. Upon hybridization with a panel of human-hamster somatic cell hybrid DNAs, the TIMP3 gene segregated with clones containing chromosome 22. Using in situ hybridization to human metaphase chromosomes, we have assigned the locus for the TIMP3 gene to the q12.1-q13.2 region of human chromosome 22.

Type IX collagen, a member of the FACIT family of extracellular matrix proteins, is a heterotrimer composed of three genetically distinct alpha chains. The cDNAs for the human and mouse alpha 1 (IX) chains have been cloned. In this paper we confirm the mapping of the human COL9A1 gene to chromosome 6q12-q13 by fluorescence in situ hybridization utilizing two genomic clones which also contain short tandem repeat polymorphisms. We also report the characterization of these repeats and their incorporation into the chromosome 6 linkage map. The COL9A1 locus shows no recombination with the marker D6Z1 (Z = 27.61 at theta = O) and identifies the most likely locus order of KRAS1P-[D6Z1-COL9A1]-D6S30. In addition, using an interspecific backcross panel, we have mapped murine Col9a1 to mouse chromosome 1. Together with other comparative mapping results, these data suggest that the pericentric region of human chromosome 6 is homologous to the most proximal segment of mouse chromosome 1. These data may facilitate linkage studies with COL9A1 (or Col9a1) as a candidate gene for hereditary chondrodysplasias and osteoarthritis.

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.

Type XI collagen is a minor and poorly characterized structural component of cartilage. Recently, cDNA and genomic clones coding for the pro alpha 1 chain of human Type XI collagen, formerly 1 alpha collagen, have been isolated and fully characterized. Here we have used one such probe to establish the chromosomal localization of the pro alpha 1 (XI) collagen gene (COL11A1) by hybridization to filter-bound DNA isolated from flow-sorted chromosomes and by in situ hybridization on metaphase chromosomes. This combination of approaches has enabled us to locate COL1A11 in the p21 region of chromosome 1. This represents the first mapping of a Type XI collagen gene and the first assignment of a collagen locus to chromosome 1. These studies also provide additional evidence for the nearly uniform dispersion of the human fibrillar collagen genes in the human genome.