Complementary physical and genetic techniques map the vinculin (VCL) gene on chromosome 10q.

Vinculin is a cytoskeletal protein component of adherens type cell junctions. The gene had been mapped to 10q11.2-qter. We have used a combination of physical and genetic mapping techniques to refine this localization. Hybridization of the vinculin cDNA probe, HV1, to a human-rodent somatic hybrid panel initially suggested a position of either 10q11.2 or 10q22.1-10q23. Genetic recombination mapping in three-generation families with multiple endocrine neoplasia type 2 (MEN2) indicated a position distal to D10S22 (10q21.1) in 10q22.1-10q23. This was confirmed by hybridization of the vinculin cDNA to flow-sorted translocation derivative chromosomes containing the q21-qter portion of chromosome 10. We conclude that the vinculin locus maps in 10q22.1-q23, distal to D10S22.     

Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA.

Previous studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7 (Guo, W., Mulligan, G. J., Wormsley, S., and Helfman, D. M. (1991) Genes & Dev. 5, 2095-2106). Using an RNA mobility-shift assay we have identified factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage. Here we present the purification to apparent homogeneity of a protein that exhibits these sequence specific RNA binding properties. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: 1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and 2) the intron regulatory element that is involved in the repression of exon 7. Our results demonstrate that the sequence requirements for PTB binding are different than previously reported and shows that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites.     

Immune complex-induced lung and dermal vascular injury. Differing requirements for tumor necrosis factor-alpha and IL-1.

Vascular injury has been induced in rat lung and dermis after deposition of IgG immune complexes (BSA-anti-BSA complexes). By the use of antibodies to TNF-alpha and IL-1 and employment of the IL-1R antagonist, the requirements for these cytokines have been evaluated. In lung, both TNF-alpha and IL-1 were required for the full expression of injury. Protection was related to the dose of cytokine-blocking agent employed and was directly correlated with diminished tissue content of myeloperoxidase (MPO). In the dermis, IL-1 was required for the full expression of injury; blocking of IL-1 protected the tissue from injury in a manner that correlated with reduced MPO content. However, anti-TNF-alpha provided no protection against dermal vascular injury and failed to reduce MPO content. In contrast, the local injection of either TNF-alpha or IL-1 beta enhanced IgG immune complex-induced dermal vascular injury, proportional to the increased tissue content of MPO, indicating that the rat dermis is reactive to both cytokines. By the employment of immunohistochemical approaches, it was demonstrated that, after deposition of immune complexes, TNF-alpha and IL-1 were readily demonstrated in lung macrophages, whereas in the dermis IL-1, but not TNF-alpha, was present in a granular pattern within interstitial cells. The immunohistochemical data are consistent with the patterns of protective effects of anti-IL-1, IL-1R antagonist and anti-TNF-alpha in the two organs. As expected, blocking of TNF-alpha or IL-1 had no protective effects on acute lung injury produced by systemic C activation after i.v. infusion of the cobra venom factor. The data suggest fundamental differences in the requirements for cytokines in lung and dermal vascular injury after deposition of IgG immune complexes.     

Polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial rRNA genes from yellowtail rockfish

A 1600 base pair region of the mitochondrial rRNA genes from 74 yellowtail rockfish, collected from three north-east Pacific localities, was amplified via PCR. RFLP analysis yielded a total of 33 restriction sites allowing examination of 139 base pairs or 0·8% of the mtDNA molecule. Essentially no variation was detected within or among the three populations.     

Fine-scale molecular genetic (RFLP) and physical mapping of a 8.9 cM region on the top arm of Arabidopsis chromosome 5 encompassing the male sterility gene, ms1

Fine-scale molecular mapping has been conducted using 183 recombinants between the markers lutescens ( lu; 17.6 cM) and transparent testa glabra ( ttg; 35.5 cM) on the top arm of Arabidopsis thaliana chromosome 5. This region contains a number of genes involved in floral development including Ms1 , a gene required for the post-meiotic development of pollen. In homozygous ms1 mutant plants, pollen development is aborted soon after microspore release, regardless of environmental conditions. The ms1 mutation is located at 29.8 ± 0.8 cM on chromosome 5. Markers have been identified which co-segregate with ms1 and should lie within 39 kb of the gene. The fine-scale map of the lu-ms1-ttg region that has been generated is significantly different from the published integrated map and provides substantially more accurate and higher marker density than the current recombinant inbred map for this region. Using clones derived from four yeast artificial chromosome libraries, a contig has been established between the RFLP markers 4111 and 4556, which encompasses the ms1 gene. This covers a genetic distance of 8.9 cM which corresponds to a physical distance of approximately 1.44 Mb, representing about 1.5–2.0% of the Arabidopsis genome. In this region, 1 cM represents a physical distance of approximately 160 kb.     

Transport and metabolism of vitamin B6 in lactic acid bacteria.

Streptococcus faecalis 8043 concentrates extracellular [3H]pyridoxal or [3H]pyridoxamine primarily as the corresponding 5'-phosphates. Accumulation of pyridoxamine requires an exogenous energy source and is inhibited by glycolysis inhibitors. A membrane potential is not required for transport of pyridoxamine, and an artificially generated potential does not drive uptake in this organism. Based on this and other evidence, it is concluded that S. faecalis accumulates pyridoxamine by facilitated diffusion in conjunction with trapping by pyridoxal kinase. Pyridoxamine-P is not concentrated, but equilibrates with that provided externally. Lactobacillus casei 7469 concentrates radioactivity only from pyridoxal, which appears internally as pyridoxal-P, suggesting that it too absorbs the vitamin by facilitated diffusion plus trapping. The specificity of the growth requirement of S. faecalis and L. casei for vitamin B6 parallels the specificity of the transport systems for this vitamin in these organisms. Lactobacillus delbrueckii 7469, however, which specifically requires pyridoxamine-P or pyridoxal-P for growth, accumulates both these compounds and pyridoxine-P from the medium, apparently by active transport, but not pyridoxine, pyridoxamine, or pyridoxal. While pyridoxal-P and pyridoxamine-P are interconvertible in this organism, pyridoxine-P is not further metabolized, thus accounting for the specificity of the growth requirement. These and previous results show (a) that different organisms may employ quite different transport machinery in utilization of a given external nutrient, and (b) that the specificity of the growth requirement for a given form of a vitamin frequently arises from the specificity of transport, but that internal metabolism of the compounds also plays a significant role in some organisms.     

Transport and metabolism of vitamin B6 in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 concentrates radioactivity intracellularly from [3H]pyridoxal or [3H]pyridoxine up to 25 times the external concentration. After 1 min of uptake intracellular radioactivity is found as phosphorylated vitamin B6. The process is sensitive to temperature and is maximally active at pH 8.1, but under the conditions tested it is insensitive to monovalent cations or metabolic inhibitors, and does not require an exogenous energy source. The Km values for uptake of pyridoxine and pyridoxal are 2.0 x 10(-7) M and 1.2 x 10(-7) M, respectively; [3H]pyridoxamine is not transported. Evidence is presented for an uptake mechanism involving facilitated diffusion followed by trapping by pyridoxal kinase. S. typhimurium also appears to lack a periplasmic binding protein for vitamin B6.     

Characterization of the female sterile (1) 1304 mutant of Drosophila melanogaster: pattern of RNA metabolism in the ovary

The female sterile mutant of Drosophila melanogaster, fs(1)1304 (1-19 +/- 2), has been characterized. Our studies show that the mutation affects the organization of nucleolar material in the ovarian nurse cells and the pattern of RNA metabolism in the ovary. Autoradiographic analysis of incorporation of 3H-uridine in vivo and analysis of 3H-uridine incorporation into high molecular weight RNA in vitro suggest that RNA from the ovaries of homozygous fs flies is degraded at a higher rate than that from heterozygous fs and wild-type ovaries. It is likely that the RNA class affected is ribosomal RNA. These data are discussed in the context of the functional role for the wild-type gene allelic to fs(1)1304, and it is suggested that one of the effects of the mutation may be on the biogenesis of ribosomes that are to be stored in the oocyte.     

Retention of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein in the Endoplasmic Reticulum Does Not Redirect Virus Assembly from the Plasma Membrane

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) has been shown to redirect the site of virus assembly in polarized epithelial cells. To test whether localization of the glycoprotein exclusively to the endoplasmic reticulum (ER) could redirect virus assembly to that organelle in nonpolarized cells, an ER -retrieval signal was engineered into an epitope-tagged variant of Env. The epitope tag, attached to the C terminus of Env, did not affect the normal maturation and transport of the glycoprotein or the incorporation of Env into virions. The epitope-tagged Env was also capable of mediating syncytium formation and virus entry with a similar efficiency to that of wild-type Env. When the epitope was modified to contain a consensus K(X)KXX ER retrieval signal, however, the glycoprotein was no longer proteolytically processed into its surface and transmembrane subunits and Env could not be detected at the cell surface by biotinylation. Endoglycosidase H analysis revealed that the modified Env was not transported to the Golgi apparatus. Immunofluorescent staining patterns were also consistent with the exclusion of Env from the Golgi. As expected, cells expressing the modified Env failed to form syncytia with CD4⁺ permissive cells. Despite this tight localization of Env to the ER, when the modified Env was expressed in the context of virus, virions continued to be produced efficiently from the plasma membrane of transfected cells. However, these virions contained no detectable glycoprotein and were noninfectious. Electron microscopy revealed virus budding from the plasma membrane of these cells, but no virus was seen assembling at the ER membrane and no assembled virions were found within the cell. These results suggest that the accumulation of Env in an intracellular compartment is not sufficient to redirect the assembly of HIV Gag in nonpolarized cells.