The Aspergillus niger carbon catabolite repressor encoding gene, creA

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.     

NCL1, a novel gene for a non-essential nuclear protein in Saccharomyces cerevisiae

The nucleolar protein Nop2p is an essential gene product that is required for pre-rRNA processing and ribosome biogenesis in Saccharomyces cerevisiae (Hong, B. et al., 1997, Mol. Cell. Biol., 17, 378–388). A search for proteins similar to Nop2p identified a novel yeast gene product that also shares significant homology with the human proliferation associated nucleolar protein p120. The gene encoding this 78 kDa protein was termed NCL1 (for nuclear protein 1; corresponding to YBL024w). Ncl1p and Nop2p contain an evolutionarily conserved motif that has been termed the ‘NOL1/NOP2/fmu family signature' (NOL1 encodes p120). Epitope tagged Ncl1p was found to be localized to the nucleus, including the nucleolus, and was concentrated at the nuclear periphery. NCL1 is not essential. Strains containing a disruption of NCL1, or strains overexpressing NCL1, grow essentially identically to wildtype NCL1 strains on a number of different media and at different temperatures. Disruption of NCL1 does not affect steady-state levels of large and small ribosome subunits, monoribosomes, and polyribosomes. However, disruption of NCL1 leads to increased sensitivity to the antibiotic paromomycin.     

A fourth gene from the Candida albicans CDR family of ABC transporters

Using primers derived from a region of the Candida albicans CDR1 (Candida drug resistance) gene that is conserved in other ABC (ATP-binding cassette) transporters, a DNA fragment from a previously unknown CDR gene was obtained by polymerase chain reaction (PCR). After screening a C. albicans genomic library with this fragment as a probe, the complete CDR4 gene was isolated and sequenced. CDR4 codes for a putative ABC transporter of 1490 amino acids with a high degree of homology to Cdr1p, Cdr2p and Cdr3p from C. albicans (62, 59 and 57% amino acid sequence identity, respectively). Cdr4p has a predicted structure typical for cluster I.1 of yeast ABC transporters, characterized by two homologous halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six transmembrane helices. In contrast to the CDR1/CDR2 genes, the genetic structure of the CDR4 gene was conserved in 59 C. albicans isolates from six different patients. Northern hybridization analysis showed that the CDR4 gene was expressed in most isolates, but no correlation between CDR4 mRNA levels and the degree of fluconazole resistance of the isolates was found. In addition, a C. albicans mutant in which both copies of the CDR4 gene were disrupted by insertional mutagenesis was not hypersusceptible to fluconazole as compared to the parent strain. Unlike CDR1 and CDR2, CDR4 does not, therefore, seem to be involved in fluconazole resistance in C. albicans.     

Expression of Xfz3, a Xenopus frizzled family member, is restricted to the early nervous system

Recent advances in analyzing wnt signaling have provided evidence that frizzled proteins can function as wnt receptors. We have identified Xfz3, a Xenopus frizzled family member. The amino acid sequence is 89% identical to the product of the murine gene Mfz3, and is predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Whole-mount in situ hybridization analysis shows that expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. In addition, Xfz3 mRNA is concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and strongly reduced anteroposterior axis in a dose-dependent manner. However, it does not affect gastrulation, the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. Our expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing activity in the morphogenesis during Xenopus development.     

The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos

The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.     

Cloning and analysis of the gene encoding lectin from the acorn barnacle Megabalanus rosa

In the acorn barnacle Megabalanus rosa, two types of galactose-binding C-type lectins (BRA-2 and BRA-3) have been identified. Here, we report the isolation and characterization of BRA-2 cDNA and genomic clones. In contrast to the BRA-3 gene, which consists of four exons, BRA-2 is encoded by a single exon, implying differences in the physiological roles of the two lectins.     

Schizosaccharomyces malidevorans and Sz. octosporus homologues of Sz. pombe rad9, a gene that mediates radioresistance and cell-cycle progression

The rad9 gene of Schizosaccharomyces pombe is involved in promoting resistance to ionizing radiation and UV light, as well as regulating cell cycle progression after irradiation. We have isolated functional rad9 cognates from two other fission yeasts, Sz. malidevorans and Sz. octosporus, that can restore radioresistance and the radiation-induced G2 delay response to Sz. pombe rad9::ura4 cells. The Sz. pombe and Sz. malidevorans genes are identical at the nucleotide sequence level, which reflects their close evolutionary relationship. Each bears three introns and codes for a 47464-Da protein that contains 426 amino acids (aa). In contrast, Sz. octosporus rad9 contains five introns and codes for a 48210-Da protein that is 432-aa long. The Sz. pombe rad9 product is only 65% identical and 80% similar to the corresponding Sz. octosporus gene product. All of the strains synthesize a rad9 RNA of approx. 1.6 kb. The presence of a rad9-like gene in these yeasts suggests that the cellular process(es) mediated by rad9, and used by these organisms to increase survival and transiently delay cycling in G2 after irradiation, are conserved. The isolation, analyses and comparison of rad9 genes from different organisms should aid in elucidating the specific biological role of the corresponding protein and especially help pinpoint regions important for function.     

Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g⁻¹, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l⁻¹, resulted in a dramatic increase in volumetric yield (85,600 U l⁻¹) and global volumetric productivity (1083 U l⁻¹ h⁻¹).     

Genomic organization of mouse Dishevelled genes

We report the isolation and characterization of two new genomic loci corresponding to the mouse Dishevelled (Dvl) genes Dvl2 and Dvl3. The Dvl genes are homologs of the Drosophila dsh segment polarity gene, and are involved in the Wnt/wingless signal transduction pathway. Dvl2 and Dvl3 genomic clones were isolated from a mouse 129 strain λFIXII genomic library and have identical exon/intron organization to Dvll. All three Dishevelled genes span 15 exons and 14 introns and have a number of conserved splice junction sites.     

胡杨WINDs基因克隆及功能初步分析*

目的: WIND(WOUND INDUCED DEDIFFERENTIATION),是属于ERF/AP2 (ETHYLENE RESPONSE FACTOR/ APETALA 2)家族的一种重要转录因子,该类基因最早被发现在拟南芥中可以与乙烯响应元件GCC-BOX和脱水响应元件DRE结合,响应干旱信号和调节乙烯水平。最近的研究发现WIND基因在植物伤口信号回应、愈伤组织形成及不定芽的产生过程中也发挥了关键作用。已有的研究阐述了WIND基因在拟南芥中控制愈伤组织形成及不定芽再生的机制,但其在木本植物中的功能尚不明确,将探究WIND基因在胡杨中与伤口信号响应及不定芽再生相关的功能,同时为在分子水平上解决胡杨再生问题提供理论依据。方法: 采用基因克隆、qRT-PCR、转基因表型分析等方法研究WIND基因在胡杨外植体伤口响应和再生不定芽过程中的作用。结果: 克隆胡杨WIND家族中的基因PeWIND1和PeWIND2,发现其编码区序列长度分别为1 050 bp和1 032 bp,编码349个和343个氨基酸,亚细胞定位均在细胞核中。组织特异性分析显示PeWIND1和PeWIND2在胡杨根、茎、叶、愈伤组织中均有表达,且在愈伤组织中表达量最高。时间表达特异性显示,在经伤口刺激后的24 h内,PeWIND1和PeWIND2基因均呈现先升高后降低的表达趋势,且均在伤口刺激后1 h达到表达量峰值。转基因植株表型统计发现,过表达PeWIND1和PeWIND2基因后转基因植株不定芽再生能力增强。结论: 在胡杨叶片有伤口刺激后,PeWIND1和PeWIND2响应伤口信号,表达量先升高后降低,PeWIND1和PeWIND2能够促进杨树茎段再生不定芽。