Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

Chicken riboflavin-binding protein. cDNA sequence and homology with milk folate-binding protein

The Rd gene is expressed in the livers and oviducts of laying hens and codes for the riboflavin-binding protein (RfBP) of egg yolk and egg white. A lambda gt11 cDNA library derived from chicken oviduct poly(A)+ RNA was screened with polyclonal rabbit antiserum to chicken RfBP. Positive clones were isolated and rescreened with a mixed oligonucleotide probe corresponding to residues 20-25 of the mature protein. The largest cDNA clone (969 base pairs) was subcloned into plasmid pIBI21, and the nucleotide sequence was determined by the dideoxynucleotide method. This clone contained the entire coding region for RfBP. The published amino acid sequence of the mature protein was confirmed. In addition, the following 17-residue signal peptide was deduced: Met-Leu-Arg-Phe-Ala-Ile-Thr-Leu-Phe-Ala-Val-Ile-Thr-Ser-Ser-Thr-Cys. Unexpectedly, the nucleotide sequence codes for 2 adjacent arginine residues at the carboxyl terminus that are not observed in the mature protein. The amino acid sequence of RfBP is homologous with bovine milk folate-binding protein. Eight of the nine pairs of cysteines involved in disulfide bonds in RfBP are conserved in folate-binding protein, as are all of the tryptophan residues. Sequence identity between homologous regions of these two vitamin-binding proteins is more than 30%.     

A rapid method for the purification of supercoiled PM2 DNA by affinity chromatography on H1 histone covalently coupled to agarose.

A simple and rapid method is described for the purification of supercoiled PM2 DNA by affinity chromatography on columns of H1 histone covalently coupled to agarose. The method does not require the use of intercalating agents or ultracentrifugation procedures. Under the conditions most appropriate for purification, elution is carried out in a single step with buffered 0.7 M NaCl after the sample has been loaded onto the column in buffered 0.2 M NaCl. The DNA eluted at the higher salt concentration consists of supercoiled closed circular DNA at greater than 90% purity independently of the ratio of supercoiled to nicked circular DNA in the input mixture.     

Directly observed 15N NMR spectra of uniformly enriched proteins

The proteins cytochrome c2, cytochrome c', and ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum were enriched in 15N by growth of the organism on 15NH4Cl. The proteins were purified to homogeneity and studied by 15N NMR. Longitudinal and transverse relaxation times as well as the nuclear Overhauser effects were determined for various groups of the proteins which vary in molecular weight from 13,000 to 114,000. The values of these parameters for the amide resonances or for groups thought to be rigid were consistent with the molecular weights of the proteins. Relaxation times of the amino-terminal alpha-amino groups and the side chain nitrogen atoms of arginine and lysine were consistent with much more rapid motion. Nitrogen atoms having bound protons were generally found to be decoupled from the protons by chemical exchange. Demonstrable 1H-15N coupling was taken as an indication that exchange was hindered, either by hydrogen bonding interactions or by inaccessibility of the group to solvent. Histidine side chain nitrogen atoms, which experience a large chemical shift upon protonation/deprotonation, were often found to be broadened beyond detectability by chemical exchange and tautomerization. Strategies for improving sensitivity and for obtaining specific peak assignments are also discussed.     

Abundance of mRNAs encoding HMG1/HMG2 class high-mobility-group DNA-binding proteins are differentially regulated in cotyledons of Pharbitis nil

The abundance of an mRNA encoding an HMG1/2 protein from Pharbitis nil (HMG1) has been previously shown to be regulated by light and an endogenous rhythm in cotyledons. A second Pharbitis nil HMG cDNA (HMG2) was characterized. The sequence of HMG2 was 82% and 86% identical to HMG1 at the nucleotide and amino acid level, respectively. As with HMG1, HMG2 mRNA was detected in all vegetative tissues and was most abundant in roots. However, unlike HMG1, HMG2 mRNA abundance did not increase upon transfer of cotyledons to darkness and did not exhibit regulation by an endogenous circadian rhythm when maintained in continuous darkness over a 68 h period. Similarly, while the abundance of HMG1 mRNA during a dark period that induced photoperiodically controlled flowering was dramatically affected by brief light exposure (night break), this treatment had no effect on HMG2 mRNA abundance. Collectively, these data are consistent with a role of HMG1 in contributing to the circadian-regulated and/or dark-regulated gene expression with constitutive expression of HMG2 playing a housekeeping role in the general regulation of gene expression in Pharbitis nil cotyledons.     

Effect of the N-terminal hydrophobic sequence of hepatitis B virus surface antigen on the folding and assembly of hybrid beta-galactosidase in Escherichia coli

To investigate the mechanism of inclusion body formation and the effect of a hydrophobic sequence on the in vivo polypeptide folding, the aggregation caused by recombinant fusion beta-galactosidase in Escherichia coli was examined. Two plasmids were constructed: pTBG(H-) carried only the preS2 sequence of the hepatitis B virus surface antigen (HBsAg) in front of the beta-galactosidase gene (lacZ) while pTBG(H+) carried an additional sequence encoding the amino-terminal hydrophobic sequence of the S region of HBsAg between preS2 and lacZ. Unlike cells expressing the fusion protein not containing the hydrophobic sequence, E. coli JM109/pTBG(H+) exhibited temperature-sensitive production of beta-galactosidase. As the culture temperature increased the activity decreased dramatically. This decrease in activity was not due to a decrease in fusion polypeptide production, but rather the fusion polypeptides containing the hydrophobic sequence aggregated within the cells at high temperature. However once the fusion polypeptides folded into proper conformation at low temperature, they maintained the activity even at high temperature. The results indicate that aggregation is a consequence of incorrect folding and assembly of the polypeptides, and is not derived from the native structure. The aggregates of the pTBG(H+)-encoded fusion polypeptides did not revert to active form when the culture temperature was lowered.