A sequence-ready BAC clone contig of a 2.2-Mb segment of human chromosome 1q24.
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Human chromosomal region 1q24 encodes two cloned disease genes and lies within large genetic inclusion intervals for several disease genes that have yet to be identified. We have constructed a single bacterial artificial chromosome (BAC) clone contig that spans over 2 Mb of 1q24 and consists of 78 clones connected by 100 STSs. The average density of mapped STSs is one of the highest described for a multimegabase region of the human genome. The contig was efficiently constructed by generating STSs from clone ends, followed by library walking. Distance information was added by determining the insert sizes of all clones, and expressed sequence tags (ESTs) and genes were incorporated to create a partial transcript map of the region, providing candidate genes for local disease loci. The gene order and content of the region provide insight into ancient duplication events that have occurred on proximal 1q. The stage is now set for further elucidation of this interesting region through large-scale sequencing. (+info)
Genomic organization and localization of the human CRMP-1 gene.
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The Collapsin Response Mediator Protein-1 (CRMP-1) is a brain specific protein considered to be involved in the collapsin-induced growth cone collapse during neural development. CRMP-1 belongs to the Unc-33 gene family. Here we report the genomic structure and the localization of the human CRMP-1 gene to chromosome 4p16.1. Sequence analysis revealed that the human CRMP-1 gene consists of 14 exons. We have also established sequencing assays for all its coding exons. This should permit the rapid screening for mutations to assess CRMP-1 role in genetic disorders mapped in the 4p16.1 region. (+info)
Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of Paracoccus denitrificans.
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The polyhydroxyalkanoic acid (PHA) granule-associated 16-kDa protein (GA16 protein) of Paracoccus denitrificans was identified, and its corresponding gene was cloned and analyzed at the molecular level. The N-terminal amino acid sequence of GA16 protein revealed that its structural gene is located downstream from the PHA synthase gene (phaCPd) cloned recently (S. Ueda, T. Yabutani, A. Maehara, and T. Yamane, J. Bacteriol. 178:774-779, 1996). Gene walking around phaCPd revealed two new open reading frames (ORFs) possibly related to PHA synthesis, one of which was the phaPPd gene, encoding GA16 protein, and the other was the phaRPd gene, encoding a protein that is putatively involved in the regulation of the expression of phaPPd. Overproduction of PhaPPd was observed in Escherichia coli carrying phaPPd, but the overproduction was not observed in the presence of phaRPd. Coexpression of phaPPd and PHA biosynthesis genes in E. coli caused increases in both the number of poly-(3-hydroxybutyric acid) (PHB) granules and PHB content and caused decreases in both the size of the granules and the molecular weight of PHB. GA16 protein was considered a phasin protein. The phaRPd gene had significant similarities to stdC, a possible transcriptional factor of Comamonas testosteroni, as well as to other ORFs of unknown function previously found in other PHA-synthetic bacteria. (+info)
Breaking colinearity in the mouse HoxD complex.
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Vertebrate Hox genes are activated in a spatiotemporal sequence that reflects their clustered organization. While this colinear relationship is a property of most metazoans with an anterior to posterior polarity, the underlying molecular mechanisms are unknown. Previous work suggested that Hox genes were made progressively available for transcription in the course of gastrulation, implying the existence of an element capable of initiating a repressive conformation, subsequently relieved from the clusters sequentially. We searched for this element by combining a genomic walk with successive transgene insertions upstream of the HoxD complex followed by a series of deletions. The largest deficiency induced posterior homeotic transformations coincidentally with an earlier activation of Hoxd genes. These data suggest that a regulatory element located upstream of the complex is necessary for setting up the early pattern of Hox gene colinear activation. (+info)
The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain.
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The gene encoding a hyperthermostable type II pullulanase produced by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated. (+info)
Physical map and organization of chromosome 7 in the rice blast fungus, Magnaporthe grisea.
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The rice blast fungus Magnaporthe grisea is a highly destructive plant pathogen and one of the most important for studying various aspects of host-plant interactions. It has been widely adopted as a model organism because it is ideally suited for genetic and biological studies. To facilitate map-based cloning, chromosome walking, and genome organization studies of M. grisea, a complete physical map of chromosome 7 was constructed using a large-insert (130 kb) bacterial artificial chromosome (BAC) library. Using 147 chromosome 7-specific single-copy BAC clones and 20 RFLP markers on chromosome 7, 625 BAC clones were identified by hybridization. BAC clones were digested with HindIII, and fragments were size separated on analytical agarose gels to create DNA fingerprints. Hybridization contigs were constructed using a random cost algorithm, whereas fingerprinting contigs were constructed using the software package FPC. Results from both methods were generally in agreement, but numerous anomalies were observed. The combined data produced five robust anchored contigs after gap closure by chromosomal walking. The genetic and physical maps agreed closely. The final physical map was estimated to cover >95% of the 4.2 Mb of chromosome 7. Based on the contig maps, a minimum BAC tile containing 42 BAC clones was created, and organization of repetitive elements and expressed genes of the chromosome was investigated. (+info)
A 12-Mb complete coverage BAC contig map in human chromosome 16p13.1-p11.2.
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We have constructed a complete coverage BAC contig map that spans a 12-Mb genomic segment in the human chromosome 16p13.1-p11.2 region. The map consists of 68 previously mapped STSs and 289 BAC clones, 51 of which-corresponding to a total of 7.721 Mb of genomic DNA-have been sequenced, and provides a high resolution physical map of the region. Contigs were initially built based mainly on the analysis of STS contents and restriction fingerprint patterns of the clones. To close the gaps, probes derived from BAC clone ends were used to screen deeper BAC libraries. Clone end sequence data obtained from chromosome 16-specific BACs, as well as from public databases, were used for the identification of BACs that overlap with fully sequenced BACs by means of sequence match. This approach allowed precise alignment of clone overlaps in addition to restriction fingerprint comparison. A freehand contig drawing software tool was developed and used to manage the map data graphically and generate a real scale physical map. The map we present here is approximately 3.5 x deep and provides a minimal tiling path that covers the region in an array of contigous, overlapping BACs. (+info)
Characterization of the DNA polymerase loci of the novel porcine lymphotropic herpesviruses 1 and 2 in domestic and feral pigs.
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Two novel porcine gammaherpesviruses, porcine lymphotropic herpesviruses 1 and 2 (PLHV-1 and -2), have been detected by amplification of short DNA polymerase (DPOL) sequences from blood and spleen of domestic pigs while searching for unknown herpesviruses in pigs as possible risk factors in xenotransplantation. In the present study, the DPOL genes of the two viruses and the open reading frames (ORFs) that follow in the downstream direction were amplified by PCR-based genome walking from adaptor-ligated restriction fragment libraries of porcine spleen samples. The sequences determined for the two PLHVs exhibited a very low G+C content (37 mol%) and a marked suppression of the CpG dinucleotide frequency. The DPOL proteins encoded were 95% identical and showed a close relationship (60% identity) to the DPOL protein of a ruminant gammaherpesvirus, alcelaphine herpesvirus 1 (AlHV-1). This was confirmed by phylogenetic analyses of the conserved regions of the two PLHV DPOL proteins. The PLHV ORFs downstream of DPOL exhibited 83% identity to each other and >>50% similarity to ORF A5, the position equivalent of AlHV-1. From these data, the PLHVs can be firmly classified to the subfamily Gammaherpesvirinae: To find a natural reservoir for the PLHVs, organs of feral pigs were screened with five different PCR assays, targetting either the DPOL gene or 3'-flanking sequences. In all samples, PLHV sequences were detected that originated predominantly from PLHV-2, suggesting the possibility of virus transfer between feral and domestic pig populations. (+info)