Characterization of the postacrosomal sheath of bovine spermatozoa

A purified head fraction was prepared from bovine epididymal spermatozoa and was utilized to identify the solubility characteristics and major polypeptide components of the postacrosomal sheath. Sperm heads extracted in nonionic-detergent-containing or high-salt-containing solutions retained an intact postacrosomal sheath, but it was readily solubilized by high pH extraction solutions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major polypeptide of 58,000 daltons (58-kD) in the high pH extract solution. Antibodies to the 58-kD polypeptide specifically reacted with the postacrosomal segment by immunofluorescence and by electron microscopic immunohistochemistry were shown to bind the postacrosomal sheath. We conclude that this 58-kD polypeptide is a constituent of the postacrosomal sheath and that its distribution is restricted to the postacrosomal segment.     

Quantitative autoradiographic analysis of [I]-human CGRP binding sites in the dorsal horn of rat following chronic constriction injury or dorsal rhizotomy

Quantitative receptor autoradiography was used to examine the binding of [¹²⁵I]-human CGRP in the dorsal horn of the L4 spinal segment of rats with a chronic constriction injury (CCI) of the sciatic nerve or unilateral dorsal rhizotomies of spinal segments L1–L6. At the times selected for study, we found no change in the amount of CGRP binding in any areas examined following CCI. In contrast, our results showed a temporally related increase in the amount of CGRP binding in areas within laminae I–II and in lateral lamina V of the dorsal horn ipsilateral to the rhizotomies. These results indicate that CGRP binding sites are regulated, most likely, by changes in the release of CGRP. Further, our results suggest that the release of CGRP from primary afferent neurons is unchanged in animals with a CCI.     

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.     

PLASTID DEVELOPMENT IN IOJAP- AND CHLOROPLAST MUTATOR-AFFECTED MAIZE PLANTS

Plastids affected by either iojap or chloroplast mutator fail to green, and altered plastids are maternally transmitted to subsequent generations. The ultrastructure of iojap-affected plastids indicates that these plastids contain no ribosomes and are capable of supporting little internal membrane organization in either light or dark-grown plants. Chloroplast mutator-affected plastids of light-grown plants contain some organized internal membrane structures. In dark-grown plants, chloroplast mutator-aftected plastids contain a crystalline prolamellar body, numerous vesicles, and osmiophilic granules. The chloroplast mutator-affecled etioplasts display an abnormal distribution of lamellar membranes; these membranes, rather than radiating in a spokelike pattern from the prolamellar body, are condensed into a portion of the organelle. Light causes disruption of the prolamellar body in chloroplast mutator-affected plastids without promoting the organization of a normal thylakoid membrane system. The effects of iojap and chloroplast mutator are cell autonomous and apparently influence the individual plastid, as evidenced by the persistence of heteroplastidic cells containing normal and affected plastids.     

EVIDENCE FOR THE OCCURRENCE AND DISTRIBUTION OF INORGANIC POLYPHOSPHATES IN VERTEBRATE TISSUES

Evidence for the occurrence of polyphosphates having apparent chain-lengths ranging from less than 10 to over 5000 orthophosphate units has been found in adult brain as well as in a number of other mammalian tissues which have been examined. There appears to be three times as much polyphosphate in rat brain as there is in rat liver. Adult rat brain appears to contain at least 15 μg of phosphorus as polyphosphate per g of fresh tissue. In addition, neural polyphosphate is extremely labile to catabolic degradation after death whereas hepatic polyphosphate is relatively more stable. The nature of this inorganic polymer was elucidated through use of the technique of ³¹P nuclear magnetic resonance spectroscopy. Further structural evidence was obtained by application of the isotopic dilution technique to ³²P-labelled neural polyphosphate mixed with an abiotically prepared inorganic polyphosphate. The conditions and rates of hydrolysis of the biological polyphosphate and a non-biological polyphosphate were comparable.