Identification of the gene encoding DNA topoisomerase I from Candida albicans

Abstract A gene encoding a type I topoisomerase (TOP1) was isolated from Candida albicans , sequenced, and expressed in Saccharomyces cerevisiae . The TOP1 gene was identified from a C. albicans genomic library by hybridization with the product of a polymerase chain reaction with degenerate primer sets encoding regions conserved in other TOP1 genes. A clone containing an open reading frame of 2463 bp and predicted to encode a protein of 778 amino acids with sequence similarity to eukaryotic type I topoisomerases was identified. The C. albicans TOP1 gene restored camptothecin sensitivity and increased the topoisomerase activity in S. cerevisiae , indicating that the DNA fragment encodes a functional C. albicans topoisomerase I.     

A Comprehensive Analysis of the Phylogeny,Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis,an Endangered Reptile Species

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 C_λ genes, each preceded by a J_λ gene, and 86 potentially functional V_λ genes upstream of (J_λ-C_λ)_n. The Igκ locus contains a single C_κ gene, 6 J_κs and 62 functional V_κs. All V_L genes are classified into a total of 31 families: 19 V_λ families and 12 V_κ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed V_λ and V_κ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.     

Functional characterization of Caenorhabditis elegans DNA topoisomerase IIIalpha

To investigate the function of a DNA topoisomerase III enzyme in Caenorhabditis elegans, the full-length cDNA of C.elegans DNA topoisomerase IIIα was cloned. The deduced amino acid sequence exhibited identities of 48 and 39% with those of human DNA topoisomerase IIIα and Saccharomyces cerevisiae DNA topoisomerase III, respectively. The overexpressed polypeptide showed an optimal activity for removing negative DNA supercoils at a relatively high temperature of 52–57°C, which is similar to the optimum temperatures of other eukaryotic DNA topoisomerase III enzymes. When topoisomerase IIIα expression was interfered with by a cognate double-stranded RNA injection, pleiotropic phenotypes with abnormalities in germ cell proliferation, oogenesis and embryogenesis appeared. These phenotypes were well correlated with mRNA expression localized in the meiotic cells of gonad and early embryonic cells.     

Genomic clones of a wild-type allele and a transposable element-induced mutant allele of the sucrose synthase gene of Zea mays L

In an attempt to isolate the transposable genetic element Ds from Zea mays L., we cloned DNA fragments hybridizing to a cDNA clone derived from the sucrose synthase gene in a λ vector (λ::Zm Sh). The fragments cloned from wild-type and from the Ds-induced mutant sh-m5933 (λ::Zm sh-m5933) share a segment 6 kb long while a contiguous segment of 15 kb of λ::Zm sh-m5933 (mutant-derived DNA) does not hybridize to the DNA segment cloned from the wild-type. Restriction maps are given, and the junction point between the two DNA segments in the mutant clone was determined. Hybridization of DNA fragments, present in the wild-type DNA of λ::Zm Sh, but not in the mutant clone, λ::Zm sh-m5933, to genomic DNA of sh-m5933 showed that no part of this DNA is deleted. It cannot be said whether the DNA found in the mutant, but not in the wild-type clone, has been brought there by Ds insertion or by another Ds-dependent DNA rearrangement. The mutant-derived DNA was hybridized to genomic DNA of various maize lines digested by several restriction endonucleases. Approximately 40 bands were detected. The mutant-derived DNA contains two pairs of inverted repeats several hundred nucleotide pairs long, one of which is located at the junction to wild-type-derived DNA.     

The topoisomerase I gene from Ustilago maydis: sequence, disruption and mutant phenotype.

The Ustilago maydis genomic TOP1 gene encoding DNA topoisomerase I was cloned by amplifying a gene fragment using the polymerase chain reaction, and using this fragment to search a genomic DNA library by hybridization. The predicted peptide sequence exhibited 30-40% identity to other eukaryotic TOP1 genes, yet differed in several features. First, an unusually long acidic region was identified near the amino terminus (28/29 residues are acidic), which resembles other nucleolar peptide motifs. Second, an atypical carboxy-terminal 'tail', absent in other TOP1 genes, followed the active site tyrosine residue. A top1 gene disruption mutant was constructed by replacing the genomic TOP1 gene, with a top1::HygR null allele. This mutant lost the abundant topoisomerase I activity evident in wild-type U.maydis, and displayed a subtle coloration phenotype evident during cell senescence.     

Characterization of cDNA encoding the mouse DNA topoisomerase II that can complement the budding yeast top2 mutation.

Several cDNA clones encoding mouse DNA topoisomerase II were obtained from a mouse spermatocyte cDNA library and the entire coding sequence of the gene was determined. The mouse DNA topoisomerase II consists of 1528 amino acids with a molecular weight of 173 kDa. It shares significant homologies with the other eucaryotic enzymes, although species-specific sequences are observed in their highly charged C-terminal regions. The complete mouse TOP2 cDNA was put under yeast GAL1 promoter and examined for complementation of top2ts mutation in S.cerevisiae. We found that the cloned mouse gene could rescue the temperature-sensitive top2ts mutation, depending on its induction by galactose. The functional expression of the mouse DNA topoisomerase II in yeast was further confirmed by enzymatic assays and by immunological methods with antibodies specific for the mouse enzyme.     

Analysis of Mice with Single and Multiple Copies of Transgenes Reveals a Novel Arrangement for the λ5-VpreB1 Locus Control Region

The murine λ5-V_preB1 locus encodes two proteins that form part of the pre-B-cell receptor and play a key role in B-lymphocyte development. We have identified a locus control region (LCR) which is responsible for coordinate activation of both genes in pre-B cells. Analysis of mice with single and multiple copies of transgenes shows a clear difference in the expression behavior of the genes depending on the transgene copy number. While expression of both λ5 and V_preB1 in single- and two-copy integrations requires the presence of a set of DNase I hypersensitive sites located 3′ of the λ5 gene, small fragments containing the genes have LCR activity when arranged in multiple-copy tandem arrays, indicating that additional components of the LCR are located within or close to the genes. The complete LCR is capable of driving efficient copy-dependent expression of a λ5 gene in pre-B cells even when it is integrated into centomeric γ-satellite DNA. The finding that activation of expression of the locus by positively acting factors is fully dominant over the silencing effect of heterochromatin has implications for models for chromatin-mediated gene silencing during B-cell development.     

Retro-recombination screening of a mouse embryonic stem cell genomic library

Targeted gene disruption is an important tool in molecular medicine, allowing for the generation of animal models of human disease. Conventional methods of targeting vector (TV) construction are difficult and represent a rate limiting step in any targeting experiment. We previously demonstrated that bacteriophage are capable of acting as TVs directly, obviating the requirement for ‘rolling out’ plasmids from primary phage clones and thus eliminating an additional, time consuming step. We have also developed methods which facilitate the construction of TVs using recombination. In this approach, modification cassettes and point mutations are shuttled to specific sites in phage TVs using phage–plasmid recombination. Here, we report a further improvement in TV generation using a recombination screening-based approach deemed ‘retro-recombination screening’ (RRS). We demonstrate that phage vectors containing specific genomic clones can be genetically isolated from a λTK embryonic stem cell genomic library using a cycle of integrative recombination and condensation. By introducing the gam gene of bacteriophage λ into the probe plasmid it is possible to select for positive clones which have excised the plasmid, thus returning to their native conformation following purification from the library. Rapid clone isolation using the RRS protocol provides another method by which the time required for TV construction may be further reduced.     

Characterization of Three Novel Human Cadherin Genes (CDH7, CDH19, and CDH20) Clustered on Chromosome 18q22–q23 and with High Homology to Chicken Cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5′ and 3′ rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3′ RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse “cadherin-7” cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22–q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.