Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa.

Usher syndrome type IIa (OMIM 276901), an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa, maps to the long arm of human chromosome 1q41 between markers AFM268ZD1 and AFM144XF2. Three biologically important mutations in Usher syndrome type IIa patients were identified in a gene (USH2A) isolated from this critical region. The USH2A gene encodes a protein with a predicted size of 171.5 kilodaltons that has laminin epidermal growth factor and fibronectin type III motifs; these motifs are most commonly observed in proteins comprising components of the basal lamina and extracellular matrixes and in cell adhesion molecules.

Transfer of sensitivity to tumor promoters by transfection of DNA from sensitive into insensitive mouse JB6 epidermal cells.

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P(+)) and nonpromotable (P(-)) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P(+) lines, JB6 Cl 41 or JB6 Cl 22, was CaPO(4) precipitated and used to transfect the P(-) cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 x 10(-8) M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P(-) JB6 Cl 30 cells yielded 40 to 50 colonies per 10(5) cells. In contrast, transfection of Cl 30 cells with "P(+) DNA" derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 10(5) cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P(+) DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P(+) lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P(+) DNA, no evidence for acquisition of promotability was obtained. P(-) JB6 Cl 25, like Cl 30, also permitted expression of transfected P(+) DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P(+) DNA and requires recipient cells of the appropriate phenotype for expression.

In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma.

The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.

Chromosomal mapping of three human LAMMER protein-kinase-encoding genes.

The eukaryotic LAMMER protein kinase family is encoded by at least three loci in the human genome, designated CLK1, 2, and 3. We have mapped these loci to 2q33, 1q21, and 15q24, respectively, by fluorescent in situ hybridization. Additionally, a CLK2 pseudo-gene has been located to 7p15-21.

Isolation of a novel human homologue of the gene coding for echinoderm microtubule-associated protein (EMAP) from the Usher syndrome type 1a locus at 14q32.

Usher syndrome type 1 (USH1) is an autosomal recessive, genetically heterogeneous disorder causing severe congenital deafness, retinitis pigmentosa, and vestibular dysfunction. The USHla locus located on 14q32 has been linked to the genetic markers D14S250 and D14S78. Using D14S250 and D14S78, we have isolated two nonchimeric YACs, 878g10 and 844g2, and a single BAC (135i20) and PAC (194e17) clone and have arranged them into a contig spanning over the D14S250 and D14S78 markers. The analysis of the YACs, BAC, and PAC revealed that the physical distance between D14S250 and D14S78 is less than 25 kb. Iterative cDNA library screening initiated with the EST 219670 found in the vicinity of the D14S78 marker yielded a cDNA contig. The nucleotide sequence of the cDNA encodes a protein of 717 amino acids in length, showing a high level of homology to the Echinoderm 77-kDa microtubule-associated protein (EMAP). The human homologue of Echinoderm microtubule-associated protein defines a novel human gene. We propose that the human EMAP is a strong candidate for the USH1a gene based on its genomic location and the proposed function of the protein.

Molecular cloning, genomic analysis, and biological properties of rat leukemia virus and the onc sequences of Rasheed rat sarcoma virus.

Rasheed rat sarcoma virus (RaSV) has been shown to code for a protein of 29,000 Mr not present in replication-competent rat type C helper virus (RaLV)-infected cells. This protein is a fused gene product consisting of a portion of the RaLV p15 gag protein and the transformation-specific 21,000 Mr (p21) ras protein, which is also found in Harvey murine sarcoma virus. We now report the molecular cloning of both the SD-1 (Sprague-Dawley) strain of RaLV and the transforming ras sequences of RaSV. Heteroduplex analysis of these cloned DNAs demonstrated that the RaSV ras gene (v-Ra-ras) was inserted into the rat type C viral genome with a small deletion of RaLV genetic information in the 5' region of the gag gene and that the v-Ra-ras gene (0.72 kilobase pair) is homologous to and colinear with the p21 ras gene of Harvey murine sarcoma virus (v-Ha-ras). Restriction enzyme mapping confirmed the homology demonstrated by heteroduplex mapping, showing strong site conservation of restriction endonucleases known to cleave v-Ha-ras. Cloned v-Ra-ras DNA transformed NIH 3T3 cells, inducing the synthesis of the p29 RaSVgag-ras protein.

Construction and characterization of a NotI linking library from human chromosome region 1q25-qter.

Chromosome 1q25-qter-specific NotI linking clones have been isolated from a NotI linking library that was constructed using DNA from MCH206.1 somatic cell hybrid cells. These cells contain chromosome 1q25-qter translocated to human chromosome Xp22 as the only human genetic material in mouse background. Sixty-eight NotI linking clones have been mapped by a combination of fluorescence in situ hybridization and R-banding to cytogenetic bands on the long arm of chromosome 1. The relative order of 11 NotI clones and their relation to known chromosome 1 markers have also been determined in 1q32 and 1q41, where the genes of Van der Woude and Usher syndrome type IIa have been previously mapped: cen-chr1.14-chr1.79-chr1.56-chr1.11-chr1.9 5- chr1.58 (chr1.74)-D1S70-chr1.15-chr1.82 (chr1.143)-chr1.62-D1S81-tel. The 1q32- and 1q41-specific NotI linking clones were sequenced in the vicinity of the NotI site. They were analyzed in terms of nucleotide composition, G+C content, frequency of CpG dinucleotides, and protein coding potentials. Most of the 1q32-q41-specific NotI linking clones were derived from CpG islands. Sequences of three NotI linking clones proved to be identical with known genes. Six of the remaining eight had a high potential for coding regions and shared short homologous regions with sequences in the GenBank database. The NotI linking clones and the identified CpG islands will provide valuable resources for constructing a long-range restriction map of chromosome 1q25-q44 and for the eventual isolation of disease genes of Van der Woude syndrome (1q32-q41) and Usher syndrome type IIa (1q41).

The genomic structure of the gene defective in Usher syndrome type Ib (MYO7A).

Usher syndrome type Ib is a recessive autosomal disorder manifested by congenital deafness, vestibular dysfunction, and progressive retinal degeneration. Mutations in the human myosin VIIa gene (MYO7A) have been reported to cause Usher type Ib. Here we report the genomic organization of MYO7A. An STS content map was determined to discover the YAC clones that would cover the critical region for Usher syndrome type Ib. Three of the YACs (802A5, 966D6, and 965F10) were subcloned into cosmids and used to assemble a preliminary cosmid contig of the critical region. Part of the gene encoding human myosin VIIa was found in the preliminary cosmid contig. A cosmid, P1, PAC, and long PCR contig that contained the entire MYO7A gene was assembled. Primers were designed from the composite cDNA sequence and used to detect intron-exon junctions by directly sequencing cosmid, P1, PAC, and genomic PCR DNA. Alternatively spliced products were transcribed from the MYO7A gene: the largest transcript (7.4 kb) contains 49 exons. The MYO7A gene is relatively large, spanning approximately 120 kb of genomic DNA on chromosome 11q13.

A yeast artificial chromosome (YAC) contig encompassing the critical region of the X-linked lymphoproliferative disease (XLP) locus.

X-linked lymphoproliferative disease (XLP) is characterized by a marked vulnerability to Epstein-Barr virus (EBV) infection. Infection of XLP patients with EBV invariably results in fatal mononucleosis, agammaglobulinemia, or malignant lymphoma. Initially the XLP gene was assigned to a 10-cM region in Xq25 between DXS42 and DXS37. Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43. In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X chromosome, or approximately 3 Mb of DNA sequence. From a human chromosome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five YACs containing DNA sequences deleted in patient 43-004 have been isolated. Sequence-tagged sites (STSs) from these YACs have been used to identify interstitial deletions in unrelated XLP patients. Three more families with interstitial deletions were found. Two of the patients (63-003 and 73-032) carried an interstitial deletion of 3.0 Mb overlapping the 43-004 deletion. In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hypogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43-004, 63-003, and 73-032. A YAC contig of 2.2 Mb spanning the XLP critical region, whose orientation on chromosome X was determined by double-color fluorescence in situ hybridization and which consists of 15 overlapping YAC clones, has been constructed. A detailed restriction enzyme map of the region has been constructed. YAC insert sizes were determined by counter-clamped homogenous electric field gel electrophoresis. Chimerism of YACs was determined by FISH and restriction mapping. On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was constructed using 5 rare-cutter restriction enzymes. The STSs and lambda subclones were used in Southern hybridization and PCR analyses. The work presented here substantially refines the critical region for XLP. The YAC contig with the overlapping interstitial deletions constitutes the basis for the construction of a transcriptional map of the critical region and facilitates the identification of the XLP gene.

The construction of a yeast artificial chromosome (YAC) contig in the vicinity of the Usher syndrome type IIa (USH2A) gene in 1q41.

The gene for Usher syndrome type II (USH2A), an autosomal recessive syndromic deafness, has been mapped to a region of 1q41 flanked proximally by D1S217 and distally by D1S439. Using sequence-tagged sites (STSs) within the region, a total of 21 yeast artificial chromosome (YAC) clones were isolated and ordered into a single contig that spans approximately 11.0 Mb. The order of microsatellite and STS markers in this region was established as D1S505-D1S425-DXS217-D1S556-D1S237-D1S4 74-EB1-EB2-KB6-AFM144XF2-KB1-K B4-D1S229-D1S490-D1S227-TGFbeta2-D1S439. Analysis of newly positioned polymorphic markers in recombinant individuals in two Usher syndrome type IIa families has enabled us to identify DXS474 and AFM144XF2 as two flanking markers for the Usher type IIa locus. The physical distance between the two markers is 1.0 Mb. This region is covered by eight YACs from the CEPH library: 945f7, 867g9, 762a6, 919h3, 794b8, 785h4, 848b9, and 841g2. A long-range physical map of the Usher type IIa critical region, using MluI, BssHII, NotI, EagI, and SacII, has been developed.