Occurrence of proteinase a isoinhibitors in wild type yeast strains and commercial baker's yeast

The occurrence of the proteinase A inhibitors 2 and 3 was investigated in wild type strains of $\text{Saccharomyces}\text{cerevisiae}$ and $\text{Saccharomyces}\text{carlsbergensis}$ as well as in several strains of commercial baker's yeast. Haploid and diploid strains of $\text{Saccharomyces}\text{cerevisiae}$ contain only proteinase A inhibitor 3 whereas in $\text{Saccharomyces}\text{carlsbergensis}$ only proteinase A inhibitor 2 is found. Strains of commercial baker's yeast contain either proteinase A inhibitor 3 or both inhibitors in a constant ratio of 1:3. Single cell cultures isolated from a strain of commercial baker's yeast also contain a mixture of the two inhibitors. Therefore, baker's yeast is not a mixture of two different cell types but the genome for both inhibitors is present in each single cell. In general, the results indicate that the occurrence of the two proteinase A inhibitors is determined genetically and, therefore, they may be called “isoinhibitors”.     

Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA.

Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV.     

Preparative separation of hemoglobins A and S by gel electrofocusing, using selective zone elution by gel transposition between suitable anolytes and catholytes.

Preparative electrofocusing on polyacrylamide gels has been limited, until recently, to excision of gel slices, diffusion, and collection of the slice diffusates. An advance was made by the introduction of a method of selective electrophoretic zone recovery by specific changes of anolyte (A. McCormick, L. E. M. Miles, and A. Chrambach, 1976, Anal. Biochem.75, 314–324). It was shown (a) that selective zone recovery could be achieved by transposition of the gels into either isoelectric ampholytes or charged buffers, (b) that it could be applied to the gram scale, and (c) that zone elution could proceed either continuously or discontinuously. The early study was, however, limited to a trivial model problem, the separation of hemoglobin from bovine serum albumin (BSA). The present study was an attempt to apply a similar selective zone recovery method to a more demanding separation problem, the separation of hemoglobin A from hemoglobin S as well as from other minor components contained in a sickle-trait human hemolysate. The study shows that selective electrophoretic zone elution from a electrofocusing gel 18 mm in diameter is capable of yielding hemoglobin A, separated from hemoglobin S, differing by only 0.2 pH units in isoelectric point. The recovery of hemoglobin A was 70%, with a load of 32 mg of hemoglobin mixture per gel, using discontinuous zone elution into a collection cup.     

Composition of splenic T lymphocytes in allophenic mice.

Guinea pig peritoneal macrophages were activated in vitro by culturing with MAF (macrophage activating factor)-containing fractions from stimulated lymphocytes. These macrophage preparations demonstrate a 60% increase in the production of prostaglandins of the E series (PGE) when compared with macrophages cultured with fractions from unstimulated lymphocytes. PGE accumulation in macrophage cultures is maximal after 24 hr with MAF; tumor cytotoxicity is also maximal at this time. The final PGE concentration in cultures of activated macrophages averaged 3 × 10^?8M.     

Transfer of memory cells into antigen-pretreated hosts. I. Functional detection of migration sites for antigen-specific B cells.

The distribution of functionally active hapten-specific B memory cells was investigated. Using antigen-pretreated lethally irradiated recipients, a marked accumulation of adoptively transferred B memory cells was demonstrated in lymph nodes containing specific antigen, but not in lymph nodes containing non-cross-reacting hapten conjugates. This difference in responsiveness between lymph nodes containing specific versus those containing nonspecific antigen developed over a period 3–5 days after memory cell transfer. The localization of antigen specific cells was T-cell independent; both carrier-primed T helper cells and specific antigenic challenge, however, were required to trigger the localized B memory cells into antibody production. Specific B memory cell accumulation did not result from an expansion of the antigen-specific cell population due to local proliferation induced by antigen depots in the lymph nodes to challenge. Rather, the results indicated that recirculating B memory cells had progressively accumulated through retention by antigen in the lymph node. These findings suggest that, in the absence of T-cell help and specific antigenic challenge, B memory cells accumulate in lymphoid tissue (follicles) without responding and provide persistent local memory for the humoral immune response.     

Stress-induced inhibition of sexual behavior: Corticosterone inhibits courtship behaviors of a male amphibian (Taricha granulosa)

When male rough-skinned newts (Taricha granulosa) are exposed to presumptive stressors, the incidence of courtship decreases and plasma corticosterone concentration increases. When sexually active males are injected intraperitoneally with corticosterone (1, 5, 10, 15, 20, or 25 μg), the incidence of courtship decreases rapidly and in proportion to the dose of corticosterone. Intracerebroventricular infusion of synthetic corticotropin-releasing factor (CRF) elevates plasma corticosterone levels and suppresses courtship. When male newts receive an injection of metyrapone, a drug that interferes with corticosterone synthesis, the inhibitory effects of stress or CRF infusion on courtship are reduced. These results support the hypothesis that, in this amphibian, elevated levels of corticosterone associated with exposure to stressful stimuli inhibit sexual behaviors.     

Mammalian cells with defective mitochondrial functions: a Chinese hamster mutant cell line lacking succinate dehydrogenase activity

A mutant cell line derived from Chinese hamster fibroblasts is described which is defective in oxidative energy metabolism. Glucose is continuously required in the medium. As a result of a block in the Krebs cycle, these cells are auxotrophs for carbon dioxide and asparagine. Several experiments support our conclusion that the mutant cells lack appreciable levels of succinate dehydorgenase activity. Other components of the electron transport chain appear to be fully functional, although there is the possibility that electron transport and oxidative phosphorylation are uncoupled.     

Topical application of B-aminopropionitrile

Application of ¹⁴CB-aminopropionitrile (BAPN) to the skin of rats followed by measurement of urinary radioactivity was used for comparative studies of the absorption of ¹⁴C-BAPN fumarate and ¹⁴C-BAPN free base (F.B.). ¹⁴C-BAPN (F.B.) was absorbed more rapidly and to a greater extent than the fumarate salt. Six hours after topical administration of ¹⁴C-BAPN (F.B.) only traces of ¹⁴C were found on the skin and less than 1% of the dose within the skin section suggesting rapid drug absorption. Lysyl oxidase activity of sponge granuloma tissue was significantly inhibited by a single small dose (20 μl) of BAPN (F.B.) applied to the intact skin over the implant. Chronic application of twice daily 5 μl doses of BAPN (F.B.) significantly inhibited lysyl oxidase of the granuloma tissue underlying the area of topical application as well as that on the opposite side not receiving BAPN. Extractibility of collagen was significantly increased only in the granuloma tissue under the skin receiving BAPN.     

Chromosomal rearrangements which affect the chromosomal integration of the ribosomal genes in Drosophila melanogaster.

Sucrose gradient analysis of DNA from detergent-pronase lysates of whole adult flies has been used to examine a variety of genotypes for the presence of ribosomal genes not integrated into the DNA of the chromosome. Such genes were found in females in which one X chromosome carries an inversion, having one of its breakpoints between the nucleolus organizer and the centromere. These inversions move the nucleolus organizer to the distal end of the X chromosome. Other inversions which do not move the nucleolus organizer, as well as a series of bobbed deficiencies, did not induce unintegrated genes. The same inversions which induce unintegrated genes in adults also produce them in the diploid brain and imaginal discs of larvae. On the other hand, in the polytene salivary glands, unintegrated genes were found in every genotype examined.     

Evidence for participation of sperm proteinases in fertilization of the solitary ascidian,Halocynthia roretzi: Effects of protease inhibitors

Effects of 15 proteinase inhibitors and an inhibitor against aminopeptidases on fertilization of the solitary ascidian, Halocynthia roretzi were studied in search of lysins. Fertilization of intact eggs was blocked by three trypsin inhibitors, leupeptin, antipain, and soybean trypsin inhibitor, and by two chymotrypsin inhibitors, chymostatin and potato proteinase inhibitor I. On the other hand, the fertilization of naked eggs was not blocked at all by leupeptin and was only partially blocked by chymostatin at the concentrations sufficient for blocking that of intact eggs. This indicates that spermatozoa utilize trypsin-like and chymotrypsin-like proteinases probably as lysins for penetrating through the chorion. The chymotrypsin-like activity appears to be also required for some step besides sperm penetration through the egg investments.     

The surfactant Bio-Solv-BBS-3 as a scintillator in liquid scintillation counting

When the surfactant mixture Bio-Solv-BBS-3 is added to a scintillation solvent it acts as a primary scintillator in response to β emissions (and Compton electrons from γs). The fluorescence excitation threshold is higher and fluorescence yield is lower than those of the primary scintillators usually employed in scintillation counting. Presence of a surfactant in a sample containing ¹⁴C or more energetic βs will be counted at higher efficiency than would be indicated by a quench correction curve (efficiency vs sample channels ratios or external standard channels ratios) derived from standards not containing surfactant.     

The origin of CSF choline and its relation to acetylcholine metabolism in brain.

Elevation of serum choline levels in the rabbit either by intravenous injection of choline or by the pharmacological action of oxotremorine results in a rise in cisternal CSF choline levels. It was excluded that the oxotremorine induced rise in CSF choline levels can be ascribed to its action on the CNS. Therefore changes in CSF choline levels can be merely the result of changes in peripheral choline stores and do not necessarily reflect changes in the cholinergic activity of the CNS. From isotopic labelling experiments the contribution of serum choline to CSF choline was found to be 42%.     

Changes in the mRNA population of chick myoblasts during myogenesis in vitro

We have analyzed the sequence complexity, frequency distribution and coding capacity of the mRNA populations of primary chick embryo muscle cultures at different stages of myogenesis. Prefusion cultures, fused myofibrillar cultures and cultures blocked for both fusion and myogenesis all contain about 17,000 different mRNA sequences, arranged in three of four abundance classes. The myofibril (96 hr) cultures, however, contain about 2500 sequences in higher concentration and six sequences in exceptionally high concentration, each present in about 15,000 copies per nucleus. These sequences are shown to be 10 times less common in premyogenic (26 hr) cultures and 40 times less common in cultures that have been blocked by BUdR against both fusion and myogenesis. The concentration of these sequences in cultures developing toward myofibril formation correlates well with the capacity of the mRNA to stimulate the cell-free synthesis of muscle-specific proteins. A more direct approach to the identity of the abundant class of myofibril mRNA indicates that it contains the templates for the synthesis of seven polypeptides that are synthesized in particularly large amounts in myogenic cultures, including myosin, actin and tropomyosin. Between 20 and 30% of the abundant mRNA is transcribed from moderately repetitive DNA sequences. The remainder of the abundant, and all of the less-abundant, mRNA is transcribed from single-copy DNA.     

Inhibitors of T-cell mitogenesis: effect of mitogen dose.

The inhibitory effects of seven compounds (hydrocortisone, histamine, interferon, isoproterenol, norepinephrine, serotonin, and γ-aminobutyric acid) were tested in phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells as a function of PHA concentration. The cultures stimulated by suboptimal levels of PHA were much more sensitive to inhibition by the added compounds, with the curve of percentage inhibition versus concentration of inhibitor shifted to the left by greater than two orders of magnitude in all cases. At the lower doses of mitogen, concentrations of hydrocortisone, histamine, interferon, and isoproterenol which approach the concentrations of these or analogous compounds in vivo caused significant and substantial inhibition of the mitogen response.     

Amino acid sequence of yeast proteinase B inhibitor 1 comparison with inhibitor 2.

The amino acid sequence of proteinase B inhibitor 1 (I^B1) from bakers' yeast has been established by automated Edman degradation up to position 42. A comparison with the sequence of proteinase B inhibitor 2 (I^B2) revealed two differences: LEU-32 and GLU-34 in I^B2 are replaced by VAL-32 and LYS-34 in I^B1. Identity of the COOH-terminal region of I^B1 with that of I^B2 was proved by degradation with the carboxypeptidases A and Y. Furthermore, a chymotryptic peptide was isolated from each of the 74 residues containing inhibitors. The two fragments, ranging from position 42 to the COOH termini of the inhibitors, were found to be identical with respect to electrophoretical mobility, end groups, amino acid composition and peptide pattern after tryptic digestion. It is concluded, that the two inhibitor sequences are identical beyond position 42. I^B1 and I^B2 are isoinhibitors, because they are coded by different genes.     

Isolation of polyribonucleotides using cellulose thin-layer plates

A thin-layer chromatographic method is described for the rapid separation and isolation of polyribonucleotides after enzymatic polymerization. This method is particularly suitable for experiments in which several variables are to be compared. The polymers prepared by this method are in yield, molecular weight, melting temperature, and template and messenger activity comparable to those isolated by other methods.