Characterization of a partial alpha-amylase clone from red porgy (Pagrus pagrus): expression during larval development.

A partial alpha-amylase cDNA was isolated from red porgy (Pagrus pagrus, Teleostei: Sparidae) and its tissue specific expression during larval development was examined. The cDNA was 949 bp long and showed 90% identity with other fish amylases. A 545 bp fragment was used to study amylase expression using in situ hybridization and RT-PCR techniques. Both methods showed a similar pattern: high and relatively constant expression for the first 30 days after hatching (dah), subsequently decreasing until the end of the experiment at 60 dah. The goal of this work was to extend the existing knowledge of the functionality of larval fish digestive systems and to provide new information about alpha-amylase gene expression.

Molecular cloning of three cDNAs that encode cysteine proteinases in the digestive gland of the American lobster (Homarus americanus).

Three clones were isolated from a lobster digestive gland cDNA library, using oligonucleotide probes based on the partial amino terminal sequence of a digestive cysteine proteinase. The cDNAs, LCP1, LCP2 and LCP3 encode preproenzymes of 322, 323 and 321 amino acid residues, and putative mature enzymes of 217, 216 and 215 residues, respectively. Calculated mature protein molecular masses are 23386 (LCP1), 29093 (LCP2) and 23255 (LCP3) Sequence alignments show that the lobster enzymes are more similar to L (55-62% identity) than H (42-44%) or B (22-24%) cathepsins. Southern analysis indicated as many as eleven genes related to the three cDNAs.

Cloning and developmental expression of a family of pleurocidin-like antimicrobial peptides from winter flounder, Pleuronectes americanus (Walbaum).

Low molecular weight antimicrobial peptides are an important component of the innate immune system in animals, yet they have not been examined widely in fish. Of particular interest is their expression during development and in response to environmental conditions and disease. Here, we report the isolation of four genomic sequences encoding putative antimicrobial peptides from the winter flounder, Pleuronectes americanus (Walbaum), as well as reverse transcription-PCR products from two tissues that form the first defensive barrier to microbes - skin and intestine. Alignment of the predicted polypeptide sequences shows a conserved hydrophobic signal peptide of 22 amino acids followed by 25 amino acids that are identical (WF2) or homologous to the amino acid sequence of pleurocidin, followed by a conserved acidic portion. Southern hybridisation analysis indicates that related peptides are encoded in the genomes of other flatfish species. Northern and RT-PCR analyses of RNA from multiple tissues show that two of the pleurocidin genes are expressed predominantly in the skin whereas two other genes are expressed mainly in the intestine. RT-PCR assays of total RNA from larvae of different ages provide the first evidence of developmental expression of antimicrobial peptides in fish and indicate that the pleurocidin gene is first expressed at 13 days post-hatch in winter flounder.

Beta-tubulins are encoded by at least four genes in the brown alga Ectocarpus variabilis.

Complementary DNA clones of two mRNA species that encode beta-tubulin in the brown alga Ectocarpus variabilis have been isolated. Sequence analysis revealed that the encoded proteins are very similar in primary structure to homologues in other eukaryotes, and differ from each other at six of 447 amino acid residues. The beta 6 message shows a preference for C- or G-terminated codons, using only 49 codons. The beta 5 message has a lesser codon bias, and makes a minor contribution to the beta-tubulin mRNA pool. Southern analysis of E. variabilis DNA demonstrated a beta-tubulin gene family of at least four members.

Increased rates of specific RNA polymerase III transcription precede overexpression of cellular oncogenes upon induction of transformation.

We have investigated a number of genes for possible overexpression upon induction of transformation, using mouse cells transformed with a temperature-sensitive mutant of simian virus 40 [SV40Ts(A)-transformed BALB-3T3, line A255], which express the transformed phenotype at 33 degrees C and suppress it as 39 degrees C. We examined both RNA polymerase II transcripts belonging to selected cellular oncogenes and RNA polymerase III transcripts of the B2 class. The selection was predicated on the basis of previous reports of an overexpression of these genes in transformed cell lines or tumors. Among the oncogenes, only cellular myc mRNA was found in increased amounts after transfer to the permissive temperature. However, nuclear run-on transcription assays indicated accelerated transcription rates for both c-myc and c-ras, with 2-3-fold and 4-5-fold increases at 2 and 6 hr post-induction, respectively. This acceleration was preceded by a 16-fold increase in the transcription rate of mouse B2 genes occurring within 30 min of the transfer to 33 degrees C. The sizeable and immediate burst in the rate of B2 transcription following the induction shift and the temporal order of activation of c-myc and c-ras are suggestive of a causal progression leading to full expression of the transformed phenotype.

Trypsinogen expression during the development of the exocrine pancreas in winter flounder (Pleuronectes americanus).

Histological, biochemical and molecular techniques were used to describe the functional development of the pancreas in winter flounder (Pleuronectes americanus) with specific reference to the expression of three trypsinogen genes. The pancreas was identified shortly following hatch, appearing as a compact structure situated dorsal and slightly posterior to the liver. As the larval fish approached metamorphosis, the pancreas became diffuse, spreading throughout the mesentery surrounding the stomach, the upper intestine and the pyloric caecae. Trypsin 2 expression was detected from 5 days post-hatch (dph). Two other related trypsinogen genes isolated from the pyloric caecae (Trypsin 1) and the intestine (Trypsin 3) showed contrasting results. Trypsin 1 showed very low levels of expression and only in late larval stages and metamorphosis. Trypsin 3 showed expression only after 20 dph. In order to determine tissue-specific expression of the three trypsinogen genes, the RNA from seven gastrointestinal-associated tissues was examined. Trypsin 1 and Trypsin 2 expression was most notably associated with the pyloric caecae, cardiac stomach, pyloric stomach and the rectum, although some variation in expression level between tissues was observed. Trypsin 3 expression had a narrower tissue distribution and was only associated with the pyloric caecae and the rectum. The tissue expression patterns observed here are likely due in part to the diffuse nature of the pancreas. Trypsin-like activity was evident from hatch and continued at significant levels through to at least 25 dph.