Richard Mazzarella

Duplication and distribution of repetitive elements and non-unique regions in the human genome

Genome mapping efforts and the initial sequencing of large segments of human DNA permit ongoing assessment of the patterns and extent of sequence duplication and divergence in the human genome. Initial sequence data indicate that the most highly repetitive sequences show isochore-related enrichment and clustering produced by successive insertional recombination and local duplication of particular repetitive elements. Regional duplication is also observed for a number of otherwise unique genomic sequences and thereby makes these segments become repetitive. The consequences of these duplication events are: (1) clustering of related genes, along with a variety of coregulatory mechanisms; and (2) recombinations between the nearby homologous sequences, which can delete genes in individuals and account for a significant fraction of human genetic disease.

Sequence and gene content in 52 kb including and centromeric to the G6PD gene in Xq28

A cosmid containing 36.4 kb of high GC human DNA centromeric to the G6PD gene has been analyzed. The sequence was 99.9% precise, based on the comparison of 4.3 kb that overlaps an earlier analysis of 20.1 kb containing G6PD. Properties of the entire 52 kb region that may be characteristic of high GC portions of the genome include a very high density of sixty-two half or full Alu sequences, or 1.2/kb, and an absence of L1 sequences. Other highly repetitive sequences include 11 MER sequences, one of them interrupted at two positions by groups of 3 Alu elements. In segments of unique sequence, computer-aided analysis predicted three possible genes, one of which has thus far been confirmed by the recovery of a corresponding cDNA, both by a direct hybridization method and by a PCR-based method based on a primer pair inferred from the genomic sequence. The cDNA has been sequenced, and is completely concordant with counterpart genomic sequence; it has no resemblance to any previously described gene.

Human protein kinase C Iota gene (PRKCI) is closely linked to the BTK gene in Xq21.3.

The human X chromosome contains many disease loci, but only a small number of X-linked genes have been cloned and characterized. One approach to finding genes in genomic DNA uses partial sequencing of random cDNAs to develop {open_quotes}expressed sequence tags{close_quotes} (ESTs). Many authors have recently reported chromosomal localization of such ESTs using hybrid panels. Twenty ESTs specific for the X chromosome have been localized to defined regions with somatic cell hybrids, and 12 of them have been physically linked to markers that detect polymorphisms. One of these ESTs, EST02087, was physically linked in a 650-kb contig to the GLA ({alpha}-galactosidase) gene involved in Fabry disease. A comparison of this contig with a 7.5-Mb YAC contig indicated that this gene is also within 250 kb of the src-like protein-tyrosine kinase BTK (X-linked agammaglobulinemia protein-tyrosine kinase) gene in Xq21.3. 14 refs., 1 fig.

Expressed STSs and transcription of human Xq28

STSs, which have been used to build and format clone contigs, have been used here to assemble a transcriptional map across a cytogenetic band. Of fifty one STSs in Xq28, 20 were positive by RT-PCR. Thus, an additional 20 possible ESTs were detected among the STSs, and seven of these also identified cDNAs in at least one library. The transcripts confirm the high expression level of this region, correlated with its GC compositional map and CpG island content.

Recombination trapping: an in-vivo approach to recover cDNAs encoded in YACs.

We have developed an approach to identify and localize cDNAs encoded by YACs. In this scheme, a YAC truncation vector containing a cDNA library is used to interrupt the YAC by homologous recombination in yeast. This approach generates YACs truncated at the site of recombination between the cDNA and the cognate YAC sequence and thus localizes the gene in the YAC. This method results in the production of a large percentage of true recombinants identifying gene encoding regions of the genome. This approach is shown to identify an unique EST sequence from a YAC in Xp22, the recently described transketolase-like gene in a YAC from Xq28 and a putative kinesin-like gene in Xq13. This system should also be useful in the mapping of YACs by targeted integration. We have constructed a new telomere truncation vector, pGR8, which incorporates two selectable markers, HIS5 and LYS2. This vector overcomes problems of previous vectors including: incompatibility with most YAC libraries, vector homology with the YAC arms and high backgrounds resulting from the use of a single selectible marker. A third counterselection with 5-fluoroorotic acid (5FOA) against yeast clones retaining the URA3 gene was also employed to reduce background further. Therefore, this vector and approach should be useful to the transcriptional analysis of YAC maps of any genome.