Molecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa.

The regulatory gene cys-3+ controls the synthesis of a number of enzymes involved in sulfur metabolism. cys-3 mutants show a multiple loss of enzymes in different pathways of sulfur metabolism. The cys-3+ gene was isolated by transformation of an aro-9 qa-2 cys-3 inl strain with a clone bank followed by screening with the "sib selection" method. The library used (pRAL1) contained inserts of Sau3a partial digest fragments of about 9 kilobases as well as the Neurospora qa-2+ gene. Double selection for qa-2+ and cys-3+ function was carried out. The transformants obtained with the isolated cys-3+ clone show recovery of the enzyme activities associated with the cys-3 mutation (e.g., arylsulfatase and sulfate permease). Restriction fragment length polymorphism experiments confirmed the identity of the clone, mRNA studies with Northern blots show that the expression of the cys-3+ gene is inducible. In contrast to cys-3+, the cys-3 (P22) mutant gene was not expressed at a higher level under sulfur-derepressed conditions.     

Molecular cloning and characterization of ovine homeo-box-containing genes

The sheep genome contains at least eleven homeo-boxes (hox). Using two hox-specific 36-mer oligodeoxynucleotides to screen a sheep genomic library, constructed in lambda Charon28, clones of nine of the hox were identified. Six of the hox clones were analysed by nucleotide sequencing, Southern-blot hybridization and Northern-blot analysis. Two of the hox appear to be cognates of the human Hu-1 (or mouse Hox 2.1) and the mouse Hox 1-3, while another is closely related to the mouse Hox 1-4. These results suggest that there is strong sequence conservation in the hox-containing genes of different mammals, and highlight the possible occurrence of an ubiquitous set of hox-containing genes in mammals. Northern-blot analysis of four sheep hox-containing genes indicates that they are all expressed during embryogenesis and that expression is temporally regulated allowing hierarchical-regulatory interaction. Interestingly, none of the cloned hox-containing sequences contain repetitive sequences.     

Molecular cloning and characterization of mutant and wild-type human beta-actin genes.

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.     

家蚕生物钟基因Bmcryl与Bmcry2的克隆及生物信息学分析

隐花色素基因(cryptochrome gene,Cry)是已确认的主要生物钟基因之一,它广泛分布于细菌和真核生物中.昆虫Cry基因分为Cry1,和Cry2两类,果蝇只有Cry1,蜜蜂等膜翅目昆虫只有Cry2.为了研究鳞翅目模式昆虫家蚕Bombyx mori的昼夜生物钟分子调控机制和昆虫CRY蛋白的进化,本研究克隆了家...     

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.     

Molecular mechanisms of entrainment in the Neurospora circadian clock

Light and temperature are 2 of the most important environmental influences on all circadian clocks, and Neurospora provides an excellent system for understanding their effects. Progress made in the past decade has led to a basic molecular understanding of how the Neurospora clock works and how environmental factors influence it. The purpose of this review is to summarize what we currently know about the molecular mechanism of light and temperature entrainment in Neurospora.     

Molecular cloning and analysis of the genes encoding the 4-hydroxyphenylacetate hydroxylase from Klebsiella pneumoniae

The Klebsiella pneumoniae genes encoding the hydroxylase involved in the meta-cleavage pathway of 4-hydroxyphenylacetic acid (4-HPA) were cloned, and the DNA fragment from the region essential for hydroxylase activity was sequenced. K. pneumoniae 4-HPA hydroxylase was composed of two proteins (HpaA and HpaH) with different molecular masses. HpaA seems to be a flavin-containing hydroxylase with a molecular mass of 58,781 Da. HpaH, with a molecular mass of 18,680 Da, seems to be a “helper” protein required for productive hydroxylation of the substrate. The hpa genes were expressed and the hydroxylase was active in Escherichia coli. Comparison of the enzyme with other monooxygenases indicates that K. pneumoniae 4-HPA hydroxylase is a member of a new family of hydroxylases. Received: 21 August 1996 / Accepted: 6 December 1996     

Molecular cloning and characterization of homeo-box-containing genes from Atlantic salmon

As the most primitive group among vertebrates, fish might serve as a model system when studying the genetic regulation of embryogenesis in higher animals. To identify genes important for early development, we have constructed a genomic library from Atlantic salmon (Salmo salar) and screened it with homeobox-containing probes from Drosophila melanogaster. Five different salmon homeoboxes were isolated. Two of these were located in the same clone, separated by only 7.5 kb. This demonstrates the presence of clustered homeobox genes in fish. The two clustered homeoboxes were sequenced and shown to be closely related to the ANT-C/BX-C class of Drosophila, being about 80% homologous to the Ultrabithorax gene (Ubx) homeobox. One of the clustered genes appears to be the salmon equivalent of the mouse Hox-2.1 gene, indicating that some of the vertebrate homeobox-containing genes are conserved in evolution. A more diverged homeobox that shares only 60% homology with Ubx, was also sequenced. In analogy to Drosophila, therefore, the salmon genome contains more than one class of homeoboxes. In addition, Northern-blot experiments demonstrated that two of the homeobox genes are expressed in salmon embryos, suggesting their importance for proper development.