Growth hormone and seawater adaptation in coho salmon, Oncorhynchus kisutch

1. Effects of growth hormone (GH) were examined on short-term aspects of seawater adaptation in coho salmon smolts. 2. Injection of somatostatin (SRIF) immediately prior to seawater entry suppressed plasma GH levels, but did not have any significant effects at 6 or 12 hr on hematocrits, plasma glucose or plasma Na+ levels. 3. Plasma GH levels increased 250% within 36 hr after seawater exposure. 4. Plasma glucose levels, in contrast, were significantly lower in the seawater fish after 36 hr post-exposure. 5. Plasma Na+ levels increased to 190 mEq/1 by 24 hr but subsequently returned to freshwater levels while hematocrits showed no significant changes over the 72 hr of exposure. 6. The significance of these results is discussed in terms of successful seawater adaptation in coho salmon.     

The effects of low pH on egg and alevin survival of kokanee and sockeye salmon, Oncorhynchus nerka

1. The effects of low pH water on embryogenesis and vitellogenesis in kokanee and sockeye salmon (Oncorhynchus nerka) were investigated. Eggs were exposed to low pH from fertilization to 45 days post-median hatch or to an episodic exposure at pH 4.0. Adult kokanee were also exposed to low pH just prior to ovulation and spawning. 2. The most sensitive stages of development during chronic or episodic exposure to low pH were early embryonic development and newly-hatched alevins. 3. Incubation of eggs at low pH caused a lower median survival, delayed hatching, higher alevin mortality and reduced the efficiency of yolk conversion to tissue of yolk-sac alevins. Those effects were more pronounced when the eggs were fertilized at low pH. 4. Exposure of sexually mature kokanee salmon to acidified water reduced egg and alevin survival, delayed embryo hatching and decreased the percent hatch. Those effects were more pronounced when their eggs were incubated at low pH.     

Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.     

Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its primary oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), are mechanism-based inhibitors of monoamine oxidases A and B. The pseudo-first-order rate constants for inactivation were determined for various analogues of MPTP and MPDP+ and the concentrations in all redox states were measured throughout the reaction. Disproportionation was observed for all the dihydropyridiniums, but non-enzymic oxidation was insignificant. The dihydropyridiniums were poor substrates for monoamine oxidase A and, consequently, inactivated the enzyme only slowly, despite partition coefficients lower than those for the tetrahydropyridines. For monoamine oxidase B, the dihydropyridiniums were more effective inactivators than the tetrahydropyridines. Substitutions in the aromatic ring had no major effect on the inactivation of monoamine oxidase B, but the 2'-ethyl- and 3'-chloro-substituted compounds were very poor mechanism-based inactivators of monoamine oxidase A. It is clear that both oxidation steps can generate the reactive species responsible for inactivation.