Gloria León

Specific alanine-tRNA species associated with fibroin biosynthesis in the posterior silk-gland of Bombyx mori L.

During the course of our study on the role and mechanism of specific tRNA changes (tRNA adaptation) during the silk-fibroin secretion phase of the posterior silk-gland development in Bombyx mori L., we undertook fractionation and purification of the preponderant tRNAs, tRNAA”, tRNAGh/, tRNAMef, tRNASer and tRNATv’, by benzoylated DEAE-cellulose column chromatography, countercurrent distribution and polyacrylamide gel electrophoresis [2111. We observed major differences in compartmentalization of isoaccepting tRNAAk’ species in the two portions of the silk-gland, the posterior and middle silk-glands. Silk-fibroin (29% alanine, 46% glycine and 12% serine) is synthesized exclusively in the posterior part, whereas sericin (6% alanine, 17% glycine and 37% serine) is produced in the middle part of silk-gland. The Al! redominant tRNAAk’ species, tRNAe and tRNA,, , in the ratio of 2 : 1 (14% and 7% of the total tRNA content, respectively) are present in the posterior silk-gland during the fibroin secretion phase of gland development, whereas only one, tRNAg, species is the main tRNAk component of the middle silk-gland and of

Prolactin gene expression and changes of prolactin pituitary level during the seasonal acclimatization of the carp

The effect of seasonal acclimatization on the extent of prolactin (PRL) gene expression and on the content of this was studied in summer- and winter-carp (Cyprinus carpio) hormone pituitary glands. PRL content in the rostral pars distalis (RPD) was evaluated by immunocytochemistry using antibodies against a cross-linked synthetic peptide comprising the sequence of 15 amino acids which conform to the primary structure of carp PRL. To assess the level of PRL gene transcription, a 24-mer synthetic oligonucleotide probe whose sequence included nucleotides 2041-2064 located in exon V of the carp PRL gene, was used. Employing in situ hybridization assays, a high expression of PRL mRNA was observed in the RPD of summer-acclimatized carp. A negligible level of transcription was observed in tissue sections of pituitary glands from winter-acclimatized carp. Concurrently, immunodetection of the PRL-producing cells in the RPD revealed that the pituitary hormone level was significantly higher in the warm season-adapted carp.

Cloning, physical mapping and genome organization of mitochondrial DNA from Cyprinus carpio oocytes

SummaryThe mitochondrial genome from Cyprinus carpio oocytes is a 10.5 megadalton, circular DNA molecule. The carp mitochondrial DNA was cloned in pBR325. Three recombinant plasmids accounted for the entire genome. Mapping of this DNA using 11 different restriction endonucleases is reported here. Both the large and small rRNA genes were then localized using Southern blot analysis. The subunit I of the cytochrome oxidase, the cytochrome b, the tRNAGlu and the URF 4 genes were localized by nucleotide sequence analysis and homology studies with human mtDNA.Our results suggest that a similar gene order has been maintained in the mitochondrial genomes of Chordata and support the hypothesis of a common ancestor for all vertebrate organelle genomes.This study constitutes the first report on the genome organization of a fish mtDNA and provides information for further investigation in connection with sequence determination, replication, and gene expression in carp mitochondria.

Specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum

To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.

Oral administration of insulin in winter-acclimatized carp (Cyprinus carpio) induces hepatic ultrastructural changes

1. The intestinal absorption of insulin in carps was assessed examining the transepithelial passage of ingested gold-labeled hormone by electron microscopy. Insulin transfer occurred mainly through the intercellular spaces between the enterocytes. 2. When reaching the lamina propria, the gold-labeled hormone gathered predominantly around the granules of the granular cells, and therefore can enter the circulatory system via the blood capillaries which are found in close contact with these cells. 3. Winter-acclimatized carp were also capable of internalizing the hormone when fed with insulin. 4. Furthermore, the absorbed hormone revealed full activity in regard to the observed changes in the ultrastructure of the liver cells of the treated cold-adapted fish. 5. The fish ingesting the hormone underwent the same type of hepatic ultrastructure reprogramming observed when winter-acclimatized carps are injected intraperitoneally with insulin, i.e. conversion to a phenotype corresponding to hepatocytes from summer-adapted carp. 6. The oral absorption of insulin by winter-acclimatized fish and its effect in reversing the cold-adaptive state might be useful for the fish culturing industry.

Carp apolipoprotein a i intestinal absorption and transfer into the systemic circulation during the acclimatization of the carp cyprinus carpio

Abstract 1. 1. Synthesis of apo A-I in the mucosa of the proximal intestine of the carp could not be measured despite the use of different experimental approaches. Nevertheless, the content of apo A-I in the intestinal tissue was identified by immunocytochemistry. 2. 2. Transepithelial apo A-I transfer from the intestinal lumen into the lamina propria was visualized in tissue sections. Summer-acclimatized fish exhibited a strong intemalization of the apolipoprotein whilst winter-acclimatized carp did not. However, the latter resembled the summer-Actapted fish on insulin treatment. 3. 3. The absorbed apo A-I concentrated in the granular cells of the lamina propria, surrounding the granules, and was released into the systemic circulation probably through blood capillaries. 4. 4. The transfer of undegraded apo A-I into the circulatory system was confirmed by using biotin-labeled carp HDL-apolipoproteins as a probe. 5. 5. The absorptive process responded to thermal Actaptation. During cold-acclimation the transfer process was negligible with respect to the warm-Actapted state. 6. 6. Apo A-I bile content was confirmed. It was suggested that at least part of the HDL used for the transport of lipids proceeds from the bile.

Heterologous mischarging as a means of tRNA fractionation. II. Isolation of E. coli tRNA1Val and tRNA1Ala

Highly purified E. coli tRNA1 Val and tRNA1 Ala have been isolated, based on the properties of heteroaminoacylated tRNAs and their behaviour on BD-cellulose chromatography.