Actin-encoding cDNAs and gene expression during the intermolt cycle of the Bermuda land crab Gecarcinus lateralis

Two actin-encoding cDNAs (act1 and act2) from Gecarcinus lateralis have been sequenced or partially sequenced and the corresponding proteins deduced. The actl cDNA has a complete ORF; the act2 cDNA lacks most of the 5′ end of the coding region. The nucleotide (nt) sequences of both clones are very similar to act sequences of many organisms, the most closely related being from another arthropod, the silkmoth Bombyx mori. The proteins Actl and Act2 are more similar to vertebrate cytoplasmic actin isoforms (β-actins) than to vertebrate muscle actins (α-actins); they are also more similar to animal actins than to those of fungi or plants. Codon usage is strongly biased toward C or G in the third position. The deduced number of amino acid (aa) residues and calculated M_r for Actl are 376 aa and 41.94 kDa, respectively. The deduced aa sequence of Actl is very similar to those of muscle actins of B. mori and Drosophila melanogaster. Southern blots indicated seven to eleven act genes in the crab genome. Northern blots probed with a segment from the 3′ UTR of actl showed a single band of approx. 1.6 kb in poly(A)⁺mRNAs from epidermis, limb bud or claw muscle and in total RNAs from ovary and gill, and two bands of approx. 1.6 and 1.8 kb in total RNA from midgut gland. Western blots of one-dimensional gels of proteins from the four layers of the exoskeleton, epidermis, limb buds and claw muscle were probed with a monoclonal Ab against chicken gizzard actin; tissue- and stage-specific changes in actin content were observed. The presence of several isoforms, and differences in their number and occurrence at various stages of the intermolt cycle, were detected on Western blots of two-dimensional gels.     

Evaluation of copper-inducible fungal laccase promoter in foreign gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The promoter region of copper-inducible laccase gene, LCC1, from Pycnoporus coccineus was explored in the heterologous expression of foreign protein in Pichia pastoris. The promoter region (P_PPLCC1) was isolated and used to replace the methanol-inducible AOXI promoter (P_AOX1) of pPICHOLI-2, an episomal expression vector for P. pastoris, to generate a new copper-inducible expression vector. The promoter activity of P_PPLCC1 was compared with those of P_AOX1 and P_CUP1, a copper-inducible promoter of a commercial vector pPICHOLI-C, using a laccase gene as a reporter gene in P. pastoris GS115. Reporter laccase activity of the culture broth reached 182 and 43 units/L for P_PPLCC1 and P_CUP1, respectively, after induction with 0.2 mM CuSO₄ at OD₆₀₀ = 1 and culture for 120 h at 15°C in complex medium containing 1% glucose. For P_AOX1 activity, yeast cells harboring P_AOX1-laccase plasmid were cultured for 120 h at 15°C in complex medium with intermittent feeding with 1% methanol every 12 h to avoid methanol toxicity. Laccase activity of culture broth was 124 units/L. Conclusively, P_PPLCC1 is a new copper-inducible promoter that shows superior performance in terms of efficiency of laccase production compared to commercial vectors. P_PPLCC1 is additionally superior to P_AOX1 since it does not require laborious feeding with a carbon source.     

Sequence, identification and effect on conjugation of the rmoA gene of plasmid R100-1

The rmoA gene was recently identified from two partially overlapping sequences corresponding to a region close to the end of the tra operon of plasmid R100. Its putative amino acid sequence showed strong homology to the Hha protein of Escherichia coli and YmoA protein of Yersinia enterocolitica, which are modulators of gene expression in response to environmental stimuli. We have cloned the rmoA gene from plasmid R100-1 in pUC19 and obtained the complete nucleotide sequence, which was previously published only partially and may have contained some mistakes. The rmoA gene product has been identified in radiolabelled minicells as a protein of the predicted molecular mass. The wild-type rmoA gene of plasmid R100-1 has been mutated by gene replacement and its effect on the efficiency of conjugation has been analysed. When grown in LB medium, cells harbouring R100-1 plasmid with a disrupted copy of rmoA showed a five-fold increase in conjugation frequency compared to cells harbouring R100-1 plasmid with the wild-type rmoA gene, grown in the same conditions. When cells were grown in NaCl-free LB medium they showed a 50-fold increase in conjugation frequency.     

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H₂O₂), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.     

A cryptic promoter in potato virus X vector interrupted plasmid construction

Background

Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2) VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation).

Results

A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR.

Conclusion

It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli) can interrupt the downstream work.     

HSM6 gene is identical to PSY4 gene in Saccharomyces cerevisiae yeasts

Previously, we isolated mutant yeasts Saccharomyces cerevisiae with an increased rate of spontaneous mutagenesis. Here, we studied the properties of HSM6 gene, the hsm6-1 mutation of which increased the frequency of UV-induced mutagenesis and decreased the level of UV-induced mitotic crossover at the region between the centromere and ADE2 gene. HSM6 gene was mapped on the left arm of chromosome II in the region where the PSY4 gene is located. The epistatic analysis has shown that the hsm6-1 mutation represents an allele of PSY4 gene. Sequencing of hsm6-1 mutant allele has revealed a frameshift mutation, which caused the Lys218Glu substitution and the generation of a stop codon in the next position. The interactions of hsm6-1 and rad52 mutations were epistatic. Our data show that the PSY4 gene plays a key role in the regulation of cell withdrawal from checkpoint induced by DNA disturbances.