Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.     

A novel molecular genetic marker for gender determination of pigeons

The absence of conspicuous sexual dimorphism in pigeons often makes it difficult to determine their sex on the basis of external morphology. We identified a novel female-specific DNA marker in pigeons, presenting the possibility of pigeon gender determination using a PCR-based method. One-hundred and twenty random primers were used for RAPD fingerprinting in order to find any sex-specific fragments in pigeons. One of these primers, OPC-20, produced a female-specific band in the DNA fingerprints. This DNA fragment was isolated from the gel and inserted into a vector for nucleotide sequencing. A novel female-specific 732 bp sequence was obtained. A pair of primers (DoveOPC20F & R) was designed, based on the cloned sequence, for amplifying the female-specific band by PCR for pigeon gender determination. Sex-specific bands in the gel were observed in all females but not in males. The PCR products in the gel were then transferred onto nylon membranes and hybridized with a DIG-labeled probe of the cloned female-specific DNA fragment. Clear hybridization signals were found only in all of the female pigeons; the same result was obtained from dot blot hybridization. This demonstrates that the sex of pigeons can be accurately and rapidly identified by PCR.     

The regulation of the yolk protein genes,a family of sex differentiation genes in Drosophila melanogaster

There are many obvious morphological and behavioural differences between male and female Drosophila, whose differing phenotypes are produced by a hierarchy of sex determination genes. These genes have been well characterised at the genetic and molecular level. Similarly, a number of sex-specific differentiation genes have been characterised, such as the chorion and vitelline membrane genes in females and the sex peptide and other accessory gland proteins in males. Despite the depth of these parallel studies, there is only one example of a direct link between the sex determination pathway and the downstream sex differentiation genes, namely the regulation of the female-specific yolk protein genes. The yolk proteins are synthesised in the fat body and ovarian follicle cells of the adult female and are subsequently transported to the oocyte where they are stored for utilization during embrygenesis. The expression of the yolk protein genes is not entirely controlled by the sex determination hierarchy, as several different regulatory pathways must interact to direct their correct sexual, temporal and spatial regulation during development.     

The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain.

The doublesex gene of Drosophila melanogaster is the final member of a well characterized hierarchy of genes that controls somatic sex determination and differentiation. The male-specific and female-specific doublesex polypeptides occupy a terminal position in the hierarchy, and thus regulate those genes responsible for the development of sexually dimorphic characteristics of the fly. To investigate the molecular mechanism by which these two related proteins interact with specific target genes, we have identified and characterized their DNA binding domains. Using gel mobility shift experiments with sequentially deleted polypeptides, site-directed mutagenesis and spectrophotometric assays, we have shown that the two doublesex proteins share a common and novel zinc finger-related DNA binding domain distinct from any reported class of zinc binding proteins. We have further shown that of 10 null dsx alleles, six encode proteins deficient in DNA binding activity, and that three of these alleles are the result of mutations that alter cysteine and histidine residues in the metal binding domain. Our results provide evidence that both the male-specific and female-specific doublesex proteins share and depend upon the same DNA binding domain for function in vivo, suggesting that both proteins bind to, but differentially regulate, a common set of genes in both sexes.     

Expression patterns of runx2, sparc, and bgp during scale regeneration in the goldfish Carassius auratus

Teleost fish scale is a dermal skeleton equipped with a strong regenerative ability. Owing to this regenerative ability, teleost fish scale can be used as a model for the regeneration of the dermal skeleton. However, there is insufficient fundamental knowledge of the regeneration, and this limits the usage of fish scale. In this study, as a first step toward understanding the molecular mechanism of the cellular differentiation during scale regeneration, we cloned the cDNAs for osteoblast-related proteins (Runx2, Sparc, and Bgp) in goldfish, and analyzed their expressions during scale regeneration. The expression profiles of these genes during scale regeneration were similar to those during mammalian osteoblastic differentiation. Specifically, runx2 expression was increased at the earliest time point, followed by sparc expression and then bgp expression. In the earlier stages, these genes were expressed in cells that formed cellular condensations and the flat cells surrounding them in the scale pocket. As the regeneration proceeded, the expressions became restricted to the episquamal, hyposquamal, and marginal scleroblasts and the cells around the marginal area of the regenerating scale. These results strongly suggest that (1) the differentiation mechanism of scleroblasts is similar to that of mammalian osteoblasts and odontoblasts, (2) scleroblast differentiation occurs around the cellular condensations at the early regeneration stage and is restricted to the marginal area of the scale at the later stage, and (3) the differentiation mechanisms are similar between the episquamal scleroblasts that produce the external layer and the hyposquamal scleroblasts that produce the basal plate.