Natural Occurrence of Left-Handed (Z) Regions in PM2 DNA

Abstract

Bacteriophage PM2 DNA, a ccc genome of high apparent superhelical density, contains left-handed (Z) regions as detected by competitive radioimmunoassay, agarose gel electrophoresis of DNA: antibody complexes and immunoelectron microscopy. The latter technique, in conjunction with partial blockage of restriction endonuclease sites by bound antibody, was used to map the left-handed regions along the DNA molecule. A cluster of four to five antibody molecules (approximately 25% of bound antibody) was located within map units 0.05–0.18 of the single Hpa II restriction site. Sequence analysis of part of this region showed the presence of several areas of high alternating purine-pyrimidine content. A strong correlation is observed between alternating pyrimidine-purine tracts of significant length and antibody binding sites.     

A nuclear specific glycoprotein representative of a unique pattern of glycosylation

Whole rat liver nuclei were reacted with UDP-[14C]galactose in the presence of bovine beta(1----4) galactosyltransferase. The reaction mixture was electrophoresed on a reducing sodium dodecyl sulfate-polyacrylamide gel. Autoradiograms of the gel demonstrated a major labeled broad band migrating with an apparent molecular weight of 65,000-66,000. A number of other less prominently labeled bands were also present. The labeled 65,000-66,000 band when cut from the gel and subjected to alkaline reduction while in the gel matrix exclusively yielded a 14C-labeled disaccharide that co-migrated with a [14C]Gal-GlcNAcol standard in descending paper chromatography. Treatment of this disaccharide with beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) from Aspergillus niger removed all the [14C]galactose label. Treatment of the labeled 65,000-66,000 polypeptide with Endoglycosidase F, however, did not remove the [14C]galactose label. Western transfer blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels performed with horseradish peroxidase-labeled succinyl wheat germ agglutinin, a lectin specific for GlcNAc, on unlabeled nuclei revealed a dominant band at 63,000-64,000. Subjecting 14C-labeled nuclei to this procedure resulted in a shift of the major horseradish peroxidase-labeled succinyl wheat germ agglutinin band to 65,000-66,000. The shifted band was coincident with the [14C]galactose band as visualized on an autoradiogram. A survey of other rat tissue nuclei revealed the same spectrum of [14C]galactose acceptor proteins with a dominant 65,000-66,000 galactose-labeled band.     

Phosphorylation of rat heart glycogen synthase: studies in cardiomyocytes and in vitro phosphorylations with cAMP-dependent kinase and protein phosphatase-1

The phosphorylation of glycogen synthase has been studied in freshly isolated adult rat cardiomyocytes. Six peaks of 32P-labeled tryptic peptides are recovered via C-18 high performance liquid chromatography (HPLC) when synthase is immunoprecipitated from 32P-labeled cardiomyocytes and digested with trypsin. When epinephrine treated cells are used as a source of enzyme, the same HPLC profile is obtained with a dramatic enhancement of 32P recovered in two of the HPLC peaks. In vitro phosphorylation of rat heart synthase by cAMP-dependent protein kinase stimulates the conversion of synthase from the I to the D form and results in the recovery of the same tryptic peptides from the C-18 as is the case for synthase derived from cardiomyocytes. Treatment of cAMP-dependent kinase phosphorylated synthase with protein phosphatase-1 leads to a reactivation of the enzyme and a dephosphorylation of the same tryptic peptides that are selectively phosphorylated in epinephrine treated cardiomyocytes. These results are discussed in relation to hormonal control of glycogen metabolism in cardiac tissue.     

Cyclic AMP, the hyperresponsiveness factor from hog kidney

The isolation and identification of a material present in the plasma of hypertensive dogs and hypertensive human patients has been under study since 1972. The earliest experiments in relation to this work, noted that plasma from hypertensive dogs cause a hyperresponse to norepinephrine when both were administered by way of the vein. Employing a rat assay system that consisted of an anesthetized rat with polyethylene catheters in the vein for giving norepinephrine and the test fractions and a catheter in the artery for blood pressure monitoring, fractions from hog kidney were tested for hyperresponsiveness activity. The active material is very comparable to cyclic AMP in molecular weight, ultraviolet spectrum, paper chromatography, Enzyme hydrolysis and activity in the anesthetized rat system. This evidence indicates that the hyperresponsiveness factor of renal origin is cyclic AMP.     

Modifications of the fertilized egg surface following the cortical reaction in Limulus polyphemus L. as viewed with the scanning electron microscope

In the development of the horseshoe crab, Limulus polyphemus, the fertilized egg undergoes a complicated cleavage (Stages 1–3) resulting in blastoderm formation (Stage 4). Stage 1 involves intralecithal cleavage and consists of nine discrete surface modifications (events) which have been briefly described with light microscopy by Brown and Barnum ('83). Since in Stage 1 the cortical reaction (events 1–4) has already been examined with ultrastructural methods, the objectives of the present study were to examine with scanning electron microscopy: (1) the first two of three intermittent granulations (events 5 and 7), and (2) the associated events characterized by smooth surfaces (events 4, 6, and 8). The first granulation occurs 2 1/2 to 3 hours after fertilization (22°C) and lasts approximately 1 1/2 hours. The second granulation appears approximately 5 hours after fertilization and lasts about 3 hours. The dynamic changes that occur during the two granulations involve the transformation of a smooth appearing embryonic surface, liberally coated with microvilli, into a granule-dominated surface on which microvilli are greatly reduced in number. Also of considerable interest are the numerous projections which begin to appear on the surface near the end of the second granulation (event 7) and dominate the surface of the following smooth step stage (event 8). Hypotheses on the significance of these dynamic changes and surface modifications involve relationships to the cell cycle, possible mechanisms for membrane storage, and secretory function.