Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

Wide distribution of a 36-megadalton plasmid among clinical and environmental Spanish isolates ofLegionella pneumophila serogroup 1

With the eventual goal of characterizingLegionella pneumophila serogroup 1 plasmids at the molecular level, we have analyzed the plasmid contents of 78 clinical and environmental Spanish isolates. After selection of a suitable alkaline lysis method, we detected plasmids with approximate molecular weights of 25, 36, 40, 61, 80, 85, 90, and 95 megadalton (MDal). Several factors (i.e., wide temporal and geographic distribution, high frequency in both clinical and environmental isolates, and apparent high copy number after subculturing) make the 36 MDal type IA plasmid an appropriate plasmid for further molecular studies.     

A new family of repetitive nucleotide sequences is restricted to the genus Zea

We have isolated a new family of moderately repetitive nucleotide sequences (about 2500 copies per haploid genome) specific to the genus Zea and absent in other graminaceous species. These sequences are interspersed in the genome and they show the same genomic organization pattern and similar copy number in all the Zea species examined. These two facts, consistency in the copy number and the same organization pattern, would indicate on the one hand that these sequences were amplified before the divergence of Zea species, and on the other hand that maize and all the teosintes could be considered as the same evolutionary population. Independent clones corresponding to the repetitive sequences have been isolated and sequenced from a genomic library of the teosinte, Zea diploperennis. The repeats, flanked by HaeIII sites, are more than 70% G + C-rich, on average 253 bp long and show 78% similarity to each other. These repetitive sequences are in a highly methylated-C context and they present some features resembling those of coding sequences, such as high CpG and low TpA content, and similar codon usage to maize genes in one of the reading frames. Moreover, the repetitive probe hybridizes with RNA extracted from different tissues of maize and from teosinte, indicating that these repeats or similar ones are present in transcribed sequences.     

The R-banding pattern of the Chinese hamster Don cell line

Chinese hamster cells (Don line) were treated in vivo with 5-BrdU and 33258-Hoechst fluorochrome for obtaining the partial inhibition of condensation that causes the R-banding pattern. Untreated chromosomes were stained by a standard G-banding method. Statistical measurements show significant differences in the band numbers between the two treatments. The Don cell line in the authors' laboratory presents some karyotypical differences from Don cell lines studied by other authors.     

Effect of some antineoplastics on metabolic heme pathway

1. The porphyrinogenic ability of several antineoplastics used in the therapy of the different cancers was evaluated. The action of cyclophosphamide, busulfan and 5-fluorouracil on the amount and nature of the accumulated hepatic porphyrins and on the activity of delta-aminolaevulinate synthase (ALA-S), were estimated at different doses and times of drug treatment in 17-day-old chick embryos. 2. It was observed that cyclophosphamide produces a significant increase in the accumulation of hepatic porphyrins at different doses as well as in the activity of the ALA-S, at all the incubation times. Cyclophosphamide alters the pattern of porphyrins accumulated in the liver, where a coproporphyrin: protoporphyrin ratio higher than in the controls can be observed. 3. Busulfan increased the hepatic porphyrins accumulated in the liver but to a lesser degree than cyclophosphamide. 4. 5-Fluorouracil did not modify the hepatic porphyrin content when it was administered at doses up to 40 mg/embryo. 5. When the embryos were injected with busulfan or 5-fluorouracil no significant differences were observed in the activity of ALA-S up to 11 hr of incubation. 6. These results indicate that cyclophosphamide has a remarkable porphyrinogenic capacity in chick embryo while busulfan, notwithstanding the fact that it alters the haem pathway, it does so to a degree that does not impair the regulation of ALA-S activity. Fluorouracil seems to be non porphyrinogenic in this system, up to 40 mg/embryo.     

Comparative studies of selective media for recovery of Staphylococcus aureus from natural waters

Several selective media currently used for the enumeration of Staphylococcus aureus from different sources were evaluated in order to establish their quantitative recovery, specificity and degree of selectivity, using different types of water samples. The highest selectivity and reliability in the enumeration of Staph. aureus from the samples was obtained on Borrego-Florido-Romero-0 (BFR-0) and KRANEP agars. The method that produced the highest recovery of Staph. aureus was BFR-0 agar with membrane filter and incubation at 36 degrees C for 48-72 h.     

Gene activity of polytene chromosomes in Drosophila species of the obscura group

The polytene chromosome puffing patterns of Drosophila guanche were established and compared with those of Drosophila subobscura. A total of 150 loci, active in some of the 17 developmental stages studied, were described and 23 of them were found to form the characteristic puffing pattern of D. guanche. Taking into account the number of puffs as well as the gene activity of each chromosome and the total gene activity, D. guanche seems to be less active than D. subobscura. Although both species show a degree of homology in their puffing patterns lower than that found for sibling species, the degree of homology is stronger than that between species belonging to the same group but to different subgroups. Thus, D. guanche and D. subobscura must be considered as phylogenetically closely related species, belonging to the same subgroup.     

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Summary A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tyl^r) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tyl^r phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyl^s) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyl^s mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyl^s mutant obtained by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tyl^r gene is not closely linked to tylosin biosynthetic genes.     

Cloning and characterization of a linear 2.3 kb mitochondrial plasmid of maize

Summary A linear 2.3 kb DNA molecule found in maize mitochondria was cloned into pUC8. A natural deletion of this plasmid, found in cmsT and some N (fertile) types of maize plants, was mapped to one end of the plasmid. A minor sequence homology to S-2, another linear mitochondrial plasmid, was detected, as well as more significant sequence homology with chloroplast and maize nuclear DNA. Hybridization to teosinte mitochondrial DNA (mtDNA) revealed the presence of part of the maize plasmid in the high molecular weight mtDNA of the maize relatives. RNA dot hybridization indicates that the plasmid is transcribed in mitochondria. The termini of the 2.3 kb linear plasmid contain inverted repeated sequences; of the first 17 nucleotides of the termini, 16 are identical to the terminal inverted repeats of the linear S plasmids found in the mitochondria of cmsS maize plants.     

Yolk polypeptide gene expression in Minute Drosophila females

Minutes have been considered for some time to be mutant at the sites of synthesis of some components of the protein synthetic apparatus. To study the hypothetical relationship between Minutes and suboptimal translation, a group of abundant proteins, the yolk polypeptides, was assayed in outcrossed females bearing M(3)w, M(3)h ^y , or M(1)n mutations. Recently emerged Minute females contained a lower amount of yolk polypeptides, in both ovarian and nonovarian tissues, than their non-Minute sisters. This low level correlated with the lower abundance of cytoplasmic RNA in Minutes compared to control females. By 1 week of age, both M(3)w and their non-Minute sibs contained the same amount of yolk polypeptides and the corresponding mRNA. The double heterozygote, ap ⁴/+;M(3)w/+, did not differ in yolk polypeptide content from control flies. M(3)w females demonstrated reduced fecundity during the period of low yolk polypeptide content but gradually increased egg deposition as yolk polypeptide levels rose. These results suggest that the low protein levels are due to the slower maturation of M(3)w, and not to less efficient translation machinery.This work was supported by the NSERC (Canada) and a Queen's University ARC grant.