Characterization of CDC45L: a gene in the 22q11.2 deletion region expressed during murine and human development.
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Amino Acid Sequence↗
Biomedical subjects
B A Roe
Publications and source records attributed to B A Roe.
Duplications on human chromosome 22 reveal a novel Ret Finger Protein-like gene family with sense and endogenous antisense transcripts.
Analysis of 600 kb of sequence encompassing the beta-prime adaptin (BAM22) gene on human chromosome 22 revealed intrachromosomal duplications within 22q12-13 resulting in three active RFPL genes, two RFPL pseudogenes, and two pseudogenes of BAM22. The genomic sequence of BAM22vartheta1 shows a remarkable similarity to that of BAM22. The cDNA sequence comparison of RFPL1, RFPL2, and RFPL3 showed 95%-96% identity between the genes, which were most similar to the Ret Finger Protein gene from human chromosome 6. The sense RFPL transcripts encode proteins with the tripartite structure, composed of RING finger, coiled-coil, and B30-2 domains, which are characteristic of the RING-B30 family. Each of these domains are thought to mediate protein-protein interactions by promoting homo- or heterodimerization. The MID1 gene on Xp22 is also a member of the RING-B30 family and is mutated in Opitz syndrome (OS). The autosomal dominant form of OS shows linkage to 22q11-q12. We detected a polymorphic protein-truncating allele of RFPL1 in 8% of the population, which was not associated with the OS phenotype. We identified 6-kb and 1.2-kb noncoding antisense mRNAs of RFPL1S and RFPL3S antisense genes, respectively. The RFPL1S and RFPL3S genes cover substantial portions of their sense counterparts, which suggests that the function of RFPL1S and RFPL3S is a post-transcriptional regulation of the sense RFPL genes. We illustrate the role of intrachromosomal duplications in the generation of RFPL genes, which were created by a series of duplications and share an ancestor with the RING-B30 domain containing genes from the major histocompatibility complex region on human chromosome 6.
Adaptor Protein Complex 1↗
PCR-based identification of postmortem microbial contaminants--a preliminary study.
Investigation of postmortem blood can reveal the presence of significant ethanol levels. However, in some instances it cannot easily be determined if the source of ethanol is from ingestion or from postmortem endogenous fermentation by contaminating microbes. Described here is a robust polymerase chain reaction (PCR)-based method for detecting the presence of common ethanol producing microbial contaminants in human blood. A set of DNA primers were designed for use in PCR to amplify and detect the genomic DNA from humans and three test microorganisms Escherichia coli, Proteus vulgaris, and Candida albicans. A rapid and reproducible protocol was developed for isolating genomic DNA from mixed human blood-microorganism samples that yields a suitable template for PCR. The organism-specific primer pairs can detect the presence of the target microorganisms in human blood at concentrations as low as 10 colony forming units/mL. The PCR products readily can be detected after agarose gel electrophoresis. This method provides an additional means of rapidly identifying microbial contaminants in postmortem blood samples.
Candida albicans↗
Comparative sequence of human and mouse BAC clones from the mnd2 region of chromosome 2p13.
The mnd2 mutation on mouse chromosome 6 produces a progressive neuromuscular disorder. To determine the gene content of the 400-kb mnd2 nonrecombinant region, we sequenced 108 kb of mouse genomic DNA and 92 kb of human genomic sequence from the corresponding region of chromosome 2p13.3. Three genes with the indicated sizes and intergenic distances were identified: D6Mm5e (>/=81 kb)-787 bp-DOK (2 kb)-845 bp-LOR2 (>/=6 kb). D6Mm5e is expressed in many tissues at very low abundance and the predicted 526-residue protein contains no known functional domains. DOK encodes the p62(dok) rasGAP binding protein involved in signal transduction. LOR2 encodes a novel lysyl oxidase-related protein of 757 amino acid residues. We describe a simple search protocol for identification of conserved internal exons in genomic sequence. Evolutionary conservation proved to be a useful criterion for distinguishing between authentic exons and artifactual products obtained by exon amplification, RT-PCR, and 5' RACE. Conserved noncoding sequence elements longer than 80 bp with >/=75% nucleotide sequence identity comprise approximately 1% of the genomic sequence in this region. Comparative analysis of this human and mouse genomic DNA sequence was an efficient method for gene identification and is independent of developmental stage or quantitative level of gene expression. [The sequence data described in this paper have been submitted to the GenBank data library under the following accession numbers: AC003061, mouse BAC clone 245c12; AC003065, human BAC clone h173(E10); AF053368, mouse Lor2 cDNA; AF084363, 108-kb contig from mouse BAC 245c12; AF084364, mouse D6Mm5e cDNA.]
Amino Acid Sequence↗
Novel sequence organization and insertion specificity of IS605 and IS606: chimaeric transposable elements of Helicobacter pylori.
IS605, an insertion sequence (IS) that is unusual in containing homologs of genes for the single putative transposases of two other unrelated IS elements (IS200 and IS1341), was found in nearly one-third of a set of 238 independent isolates of the gastric pathogen Helicobacter pylori. Hybridization and PCR tests indicated that any strain carrying one of these ORFs also carried the other, which implies that both ORFs are in the same unit of transposition. The IS605 ends and target sites for insertion were identified by sequencing eight preexisting insertions in strain NCTC11638, corresponding empty sites in other strains, and new transpositions in E. coli of an IS605 derivative marked with a selectable chloramphenicol-resistance gene. These tests showed that IS605 is also unusual in: (1) having unique, not inverted repeat, ends; (2) not duplicating (or deleting) target sequences during transposition; and (3) inserting with its left (IS200-homolog) end next to 5'-TTTAA or 5'-TTTAAC. IS605 was implicated in at least two genome rearrangements in strain NCTC11638. A second member of the IS605 family, called IS606 (25% amino acid identity to IS605 in inferred proteins) was found in one-third of 38 H. pylori strains tested, many of which did not carry IS605. The features of these two chimaeric IS elements are discussed in terms of possible transposition mechanisms, IS element evolution, and effects of IS elements on genome organization and evolution in the microbes that they inhabit.
Base Sequence↗
Characterization of the human NIPSNAP1 gene from 22q12: a member of a novel gene family.
Rapid progress in sequencing of human and other genomes allows high-resolution analysis of their gene content on the basis of comparison between species. We have used a combined computer and biochemical approach to characterize 135 kb of human genomic sequence from 22q12 and discovered a new 10 exon gene, termed NIPSNAP1, located between the neurofibromatosis type 2 and the pK1.3 genes. The NIPSNAP1 gene spans 26 kb of genomic sequence and shows to large introns in the 5'-region. All exon-intron junctions contain the gt/ag consensus splice site. The putative promoter of the NIPSNAP1 gene is TATA-less and resides in a GC-rich island characteristic of housekeeping genes. The NIPSNAP1 mRNA is 2.1 kb, is expressed ubiquitously at variable levels, with the highest expression in liver, is terminated by an uncommon ATTAAA polyadenylation site, and is capable of encoding a 284-amino-acid protein. This NIPSNAP1 protein has a strong sequence similarity limited to the central portion of a hypothetical protein (acc. P34492) from chromosome III of C. elegans, in which the other portions resemble a 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein. Thus, the NIPSNAP1 gene is a member of an evolutionarily well conserved, novel gene family with two members in human and mouse that have now been characterized, and one member in C. elegans. The second human gene, NIPSNAP2, is localized in the vicinity of marker D7S499 on chromosome 7. Although the function of the NIPSNAP protein family is unknown, clues about its role may reside in the co-expression of the C. elegans orthologue, within an operon encoding protein motifs known to be involved in vesicular transport.
Amino Acid Sequence↗
Cloning of crustacean ecdysteroid receptor and retinoid-X receptor gene homologs and elevation of retinoid-X receptor mRNA by retinoic acid.
We report the cloning and analysis of ecdysteroid receptor (bpEcR) and retinoid-X receptor (UpRXR) cDNA homologs from the fiddler crab Uca pugilator. The deduced amino acid sequence of this crustacean EcR most closely resembles the insect EcRs within the DNA binding and ligand binding domains (LBDs). For UpRXR, the DNA binding domain (DBD) shares greatest identity to the insect USPs. The ligand binding domain, however, is closer to vertebrate RXRs but may have a nonfunctional AF-2 domain. Probes derived from these clones were used to examine transcript levels in blastemas during early limb regeneration. Both UpEcR and UpRXR transcripts were detected in low amounts 1 day after limb loss, but increased during the next 4 days. Immersion of crabs in sea water containing all-trans retinoic acid increased the steady state concentrations of UpRXR transcript and altered the pattern of circulating ecdysteroids. These effects correlate with the disruptive effects of retinoic acid on blastemal differentiation observed in earlier studies.
Amino Acid Sequence↗
Characterization of the human synaptogyrin gene family.
Genomic sequencing was combined with searches of databases for identification of active genes on human chromosome 22. A cosmid from 22q13, located in the telomeric vicinity of the PDGFB (platelet-derived growth factor B-chain) gene, was fully sequenced. Using an expressed sequence tag-based approach we characterized human (SYNGR1) and mouse (Syngr1) orthologs of the previously cloned rat synaptogyrin gene (RATSYNGR1). The human SYNGR1 gene reveals three (SYNGR1a, SYNGR1b, SYNGR1c) alternative transcript forms of 4.5, 1.3 and 0.9 kb, respectively. The transcription of SYNGR1 starts from two different promoters, and leads to predicted proteins with different N- and C-terminal ends. The most abundant SYNGR1 a transcript, the 4.5-kb form, which corresponds to RATSYNGR1, is highly expressed in neurons of the central nervous system and at much lower levels in other tissues, as determined by in situ hybridization histochemistry. The levels of SYNGR1b and SYNGR1c transcripts are low and limited to heart, skeletal muscle, ovary and fetal liver. We also characterized two additional members of this novel synaptogyrin gene family in human (SYNGR2 and SYNGR3), and one in mouse (Syngr2). The human SYNGR2 gene transcript of 1.6 kb is expressed at high levels in all tissues, except brain. The 2.2-kb SYNGR3 transcript was detected in brain and placenta only. The human SYNGR2 and SYNGR3 genes were mapped by fluorescence in situ hybridization to 17qtel and 16ptel, respectively. The human SYNGR2 gene has a processed pseudogene localized in 15q11. All predicted synaptogyrin proteins contain four strongly conserved transmembrane domains, which is consistent with the M-shaped topology. The C-terminal polypeptide ends are variable in length, display a low degree of sequence similarity between family members, and are therefore likely to convey the functional specificity of each protein.
Amino Acid Sequence↗
Cloning and sequence analysis of four t(9;11) therapy-related leukemia breakpoints.
The t(9;11)(p22;q23) is the most common chromosomal translocation in topoisomerase II inhibitor therapy-related acute myeloid leukemia (tAML). This translocation fuses the MLL and AF9 proto-oncogenes producing a novel chimeric protein. In order to gain insight into the mechanism generating the t(9;11) and to clarify the role topoisomerase II inhibition may play in that mechanism we have cloned and sequenced the breakpoints from four tAML patients with the t(9;11). This sequence analysis identifies topoisomerase II consensus binding sequences near or at the chromosome 11 and chromosome 9 breakpoints in all four patients. One patient also had the consensus binding sequence for the TRANSLIN DNA-binding protein at the 9p22 and 11q23 breakpoints. Our results further support a direct role for topoisomerase II in the genesis of these tAML translocations.
Acute Disease↗
Analyses of the cag pathogenicity island of Helicobacter pylori.
Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal-type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a approximately 37kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA+ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left 'cagII' half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis ('Ptl'), Agrobacterium tumefaciens ('Vir'), and conjugative plasmids ('Tra') that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HsIU (heat-shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL-8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end-points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cag-encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.
Antigens, Bacterial↗
First report on the systematic sequencing of the small genome of Encephalitozoon cuniculi (Protozoa, Microspora): gene organization of a 4.3 kbp region on chromosome I.
Belonging to a large group of parasitic amitochondrial protozoans (Microspora), Encephalitozoon cuniculi infects humans and other mammals. Because of its medical importance and small genome size (2.9 Mbp), we are systematically sequencing its smallest (217 kbp) chromosome. The shotgun cloning strategy now has produced the sequence of randomly dispersed contigs representing more than 180 kbp of this chromosome. The present report describes analysis of the 4.3 kbp contig, which includes the complete coding regions of dihydrofolate reductase (DHFR), thymidylate synthase (TS), and serine hydroxymethyl transferase (SHMT) genes and the partial coding region of an aminopeptidase (AP) gene. In contrast to the other reported protozoan genes, DHFR and TS are encoded by two different open reading frames (ORFs). The SHMT gene is the first one identified in a protozoan and corresponds to the cytosolic form of the enzyme. No introns were detected, and the intergenic noncoding regions do not exceed 50 bp. The mean GC content is close to 60%, and there is a G or C third-base codon bias. Transcription and translation initiation signals also are analyzed, and a model for the mRNA-ssu rRNA interactions is proposed.
Amino Acid Sequence↗
The beautifully simple but intriguingly complex world of small genomes.
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Genome, Bacterial↗
Identification and molecular analysis of lbpBA, which encodes the two-component meningococcal lactoferrin receptor.
We identified lbpB, encoding the lipoprotein component of the meningococcal lactoferrin receptor. An LbpB mutant was unable to acquire Fe from lactoferrin and exhibits decreased surface binding to lactoferrin. Primer extension and reverse transcription-PCR analysis indicate that lbpB and lbpA are cotranscribed on a polycistronic Fe-repressible mRNA.
Amino Acid Sequence↗
Analysis of the alcABC operon encoding alcaligin biosynthesis enzymes in Bordetella bronchiseptica.
We previously cloned a B. bronchiseptica (Bb) genomic DNA fragment that complements a Bb alcaligin biosynthesis mutant, and reported the identification of a gene, alcA, with predicted protein sequence similarity to siderophore biosynthesis enzymes from other organisms. In the present study we show that further nt sequencing of this region revealed two open reading frames (ORFs) 3' to alcA that encode putative proteins AlcB and AlcC, with significant sequence similarity to the aerobactin biosynthesis enzymes IucB and IucC, respectively. RT-PCR analysis indicated that the three ORFs are encoded on a single transcript, and that this operon is repressed at the transcriptional level by Fe. Primer extension analysis placed the transcriptional start point (tsp) 35 nt from the 5' end of the Fur consensus sequence and 188 nt from the putative start of translation of AlcA.
Amino Acid Sequence↗
A 2.8-Mb clone contig of the multiple endocrine neoplasia type 1 (MEN1) region at 11q13.
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder that results in parathyroid, anterior pituitary, and pancreatic and duodenal endocrine tumors in affected individuals. The MEN1 locus is tightly linked to the marker PYGM on chromosome 11q13, and linkage analysis has placed the MEN1 gene within a 2-Mb interval flanked by D11S1883 and D11S449. As a step toward cloning the MEN1 gene, we have constructed a 2.8-Mb clone contig consisting of YAC and bacterial clones (PAC, BAC, and P1) for the D11S480 to D11S913 region. The bacterial clones alone represent nearly all of the 2.8-Mb contig. The contig was assembled based on a high-density STS-content analysis of 79 genomic clones (YAC, PAC, BAC, and P1) with 118 STSs. The STSs included 22 polymorphic markers and 20 transcripts, with the remainder primarily derived from the end sequences of the genomic clones. An independent cosmid contig for the 1-Mb PYGM-SEA region was also generated. Support for correctness of the 2.8-Mb contig map comes from an independent ordering of the clones by fiber-FISH. This sequence-ready contig will be a useful resource for positional cloning of MEN1 and other disease genes whose loci fall within this region.
Chromosomes, Artificial, Yeast↗
Positional cloning of the gene for multiple endocrine neoplasia-type 1.
Multiple endocrine neoplasia-type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized by tumors in parathyroids, enteropancreatic endocrine tissues, and the anterior pituitary. DNA sequencing from a previously identified minimal interval on chromosome 11q13 identified several candidate genes, one of which contained 12 different frameshift, nonsense, missense, and in-frame deletion mutations in 14 probands from 15 families. The MEN1 gene contains 10 exons and encodes a ubiquitously expressed 2.8-kilobase transcript. The predicted 610-amino acid protein product, termed menin, exhibits no apparent similarities to any previously known proteins. The identification of MEN1 will enable improved understanding of the mechanism of endocrine tumorigenesis and should facilitate early diagnosis.
Amino Acid Sequence↗
Multicellular ascomycetous fungal genomes contain more than 8000 genes.
Fungi comprise a large monophyletic group of uni- and multicellular eukaryotic organisms in which many species are of economic or medical importance. Fungal genomes are variable in size (13-42 Mb), and multicellular species support true spatial and temporal cell-type-specific regulation of gene expression. In a 38.8-kb Aspergillus nidulans contiguous genomic DNA region, a transposable element and 12 potential genes were identified, 7 similar to genes in other organisms. This observation is consistent with the prediction that multicellular ascomycetous fungi harbor 8000-9000 genes in a 36-Mb average genome. Thus, the genomic DNA sequence of filamentous fungi will provide substantial amounts of genetic and functional information that is not available in yeast, for the human and other metazoan minimal gene complement.
Animals↗