Length Polymorphisms in Human Proline-Rich Protein Genes Generated by Intragenic Unequal Crossing over

Southern blot hybridization analysis of genomic DNAs from 44 unrelated individuals revealed extensive insertion/deletion polymorphisms within the BstNI-type loci (PRB1, PRB2, PRB3 and PRB4) of the human proline-rich protein (PRP) multigene family. Ten length variants were cloned, including alleles at each of the four PRB loci, and in every case the region of length difference was localized to the tandemly repetitious third exon. DNA sequences covering the region of length variation were determined for seven of the alleles. The data indicate (1) that the PRB loci can be divided into two subtypes, PRB1 plus PRB2, and PRB3 plus PRB4, and (2) that the length differences result from different numbers of tandem repeats in the third exons. Variant chromosomes were also identified with different numbers of PRP loci resulting from homologous but unequal exchange between the PRB1 and PRB2 loci. The overall data are compatible with the observed length variants having been generated via homologous but unequal intragenic exchange. The results also indicate that these crossover events are sensitive to the amount of homology shared between the interacting DNA strands. Allelic length variants have arisen independently at least 20 times at the PRB loci, but only one has been detected at a PRH locus. Comparison of the detailed structures of the repetitious regions in PRB and PRH loci shows that the repeats in PRB genes are very similar to each other in sequence and in length. The PRH genes contain fewer repeats, which differ considerably in their individual lengths. These differences suggest that the larger number of length variants in PRB genes is related to their greater ease of homologous but unequal pairing compared to PRH genes.     

Beta-adrenergic stimulation enhances translocation, processing and synthesis of lipoprotein lipase in rat heart cells

Cells isolated from newborn rat hearts were cultured for 10-14 days, and lipoprotein lipase activity was present in an intracellular and heparin-releasable pool. Treatment of the cultures with 10(-7) M isoproterenol for 3 min resulted in a 3-fold increase in heparin-releasable lipoprotein lipase and a concomitant decrease in residual cellular enzyme activity. Similar results were obtained by treatment with dibutyryl cAMP. Treatment with isoproterenol or dibutyryl cAMP for 2 h affected glycosylation of immunoadsorbable lipoprotein lipase, so that the ratio of [3H]galactose to [14C]mannose in the heparin-releasable enzyme increased from 3.8 (control) to 13.0 (isoproterenol-treated). The change in the ratio of the sugars in the cellular fraction of the enzyme was from 3.1 to 9.9. 2 h treatment with isoproterenol did not enhance new enzyme synthesis, as determined by incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase. 24 h after addition of either isoproterenol or dibutyryl cAMP to the culture medium, stimulation of enzyme synthesis was demonstrated. The present results permit three effects of isoproterenol on lipoprotein lipase to be distinguished: stimulation of translocation from a cellular to heparin-releasable pool; enhanced processing of mannose residues and terminal glycosylation; stimulation of synthesis of enzyme protein.     

Nonlinear stiffness--force relationships in whole mammalian skeletal muscles

Small, sinusoidal length changes were superimposed on isometric contractions of fast- and slow-twitch mouse muscles, which were stimulated maximally via their nerves. Stiffness increased with increasing frequency of sinusoidal stimulation, but the relative time course of force and stiffness changes during twitch, tetanic, or partially fused contractions was quite invariant over a range of frequencies in both muscles. Typically, stiffness increases more rapidly than force during contraction and decreases less rapidly during relaxation. This pattern was observed at various temperatures and with various numbers of stimuli. It can be described by a nonlinear relation between stiffness and force with some hysteresis. The presence in the muscle of parallel and series elastic elements, whose stiffness varies with force, may account for the nonlinear relation. This nonlinearity can be used to relate the patterns for summation of force and stiffness observed with brief trains of stimuli under a variety of conditions.     

The relationship between maternal age and chromosome size in autosomal trisomy.

The pattern of maternal age-specific incidence of autosomal trisomy in spontaneous abortions was examined for each chromosome for which a sufficient number of trisomies was observed. This included chromosomes 2, 4, 7-10, 13-16, 18, and 20-22. The rate of increase after age 30 for each of the small chromosomes (groups D-G) was similar, with the exception of chromosome 16, which showed a significantly shallower rate. The C group chromosomes tended to have an intermediate rate of increase after age 30, with the exception of chromosome 7, which had a pattern similar to the smaller chromosomes. The larger chromosomes (2 and 4) had the smallest rate of increase. There was a significant relationship between chromosome size and rate of increase after age 30 (after excluding chromosome 16), but not with rate of increase before age 30. The results suggest that autosomal trisomies may be of heterogeneous origin, with a maternal age-related factor associated with chromosome size and other sources unrelated to chromosome size. Additional evidence for and against this hypothesis is discussed.     

Heterogeneity of EBV-transformable human B lymphocyte populations

Although most human B cells express receptors for Epstein Barr virus (EBV), few (usually less than 1%) are readily transformed into B lymphoblastoid cell lines after exposure to EBV. Transformable cells previously have been found to be mostly resting B lymphocytes. We recently developed a limiting dilution culture system which permits the growth of EBV-transformed B lymphocytes with high efficiency. Because in this system up to over 30% of peripheral blood- or tonsil-derived B cells respond to EBV, we re-examined the properties of EBV-transformable cells. Frequencies of transformable lymphocytes were determined by Poisson analysis. EBV-susceptible B cells committed to IgM, IgG, or IgA secretion were found to occur in the range of 3 to 27, 0.1 to 6, and 0.1 to 5 per 100 B cells, respectively. Under our culture conditions, a major proportion of the IgM-committed cells derived from large lymphocytes which appeared to have entered the cell cycle. This population contains most of the EBV-responsive cells detected and, therefore, most of the additional cells responding in our culture system. In contrast, precursors of IgG- or IgA-producing lymphoblast lines were small cells with DNA contents typical for the G0/G1 phases of the cell cycle. Anti-immunoglobulin antibodies were used in panning experiments to separate B cell subpopulations which expressed different immunoglobulin isotypes on their surface. In limiting dilution cultures of these purified B lymphocytes subsets, it was found that virtually all precursors of IgM-producing cell lines expressed surface IgM (sIgM) before their infection and transformation by EBV. The "cloning efficiency" of positively selected, large sIgM+ cells approached 100%. In contrast, sIgG or sIgA were found only on cells committed to the production of IgG or IgA, respectively. The expression of sIgD was examined by using sequential panning procedures. Virtually all IgM-committed lymphocytes and a subset of cells committed to IgA secretion were found among the sIgD+ cells. The majority of cells committed to IgA production and all IgG-committed cells were found in the sIgD- B cell population. Our findings indicate that the EBV-susceptible B cell subset of normal lymphocytes is heterogeneous with respect to cell size, cell cycle, sIg determinants, and efficiency of transformation. On the basis of our findings and previous reports, we propose a model in which transformability is a B cell-inherent property. Factors unrelated to the virus but present in our culture system appear responsible for the enhanced vulnerability to viral transformation in some cells which entered into the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective allele loss and interference between cauliflower mosaic virus DNAs

Summary Some cauliflower mosaic virus (CaMV) alleles are selectively lost during growth of the virus in mixedly infected turnip plants. Viral DNA from plants co-inoculated with DNA of the cabbage S isolate and infectious cabbage S DNA with an extra EcoRI restriciion site lacked the extra site. The EcoRI allele was also lost in most plants co-inoculated with a non-infectious mutant of cabbage S DNA while little selective allele loss was observed with two other non-infectious mutant DNAs. Plants co-inoculated with DNAs of closely-related isolates (CM4-184 and W) contained both parental viral DNAs and some DNAs with characteristics of both parents. Interference, scored as a reduced frequency of infection or a delay in symptom appearance relative to plants inoculated with wild-type DNA, occurred when plants were inoculated with wild-type and mutant DNAs covalently attached to one another in partial dimer plasmid DNAs. Similarities in the conditions leading to selective allele loss and those leading to interference suggest that both may have been due to active gene conversion between CaMV DNA molecules.     

Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells

A simple enzymatic method for the quantitation of the mass of sn-1,2-diacylglycerol (DAG) present in crude lipid extracts was developed to assess the function of DAGs as intracellular "second messengers" of extracellular agents and of oncogene products. The assay employed Escherichia coli DAG kinase which constituted approximately 15% of the membrane protein of a plasmid-bearing strain and defined mixed micellar conditions to solubilize the DAG present and allow its quantitative conversion to [32P]phosphatidic acid. The assay was proportional with the amount of DAG added over the range of 25 pmol to 25 nmol. The rapid rise of DAG in platelets stimulated with thrombin (210% over basal) and in hepatocytes stimulated with vasopressin (230% over basal) was quantitated and the values agreed with previous measurements. The amounts of DAG in normal rat kidney (NRK) cells grown at 34 and 38 degrees C, respectively, were 0.47 and 0.61 nmol/100 nmol of phospholipid. In K-ras-transformed NRK cells grown at 34 or 38 degrees C, DAG levels were elevated 168 or 138%, respectively. When a temperature-sensitive K-ras NRK cell line was investigated, the amount of DAG present was elevated at the permissive but not at the restrictive temperature. These data are consistent with the K-ras protein functioning in transmembrane signalling by activating phospholipase C. Protein kinase C (Ca2+/phospholipid-dependent enzyme) activation by DAG may play an important role in cellular transformation.     

Importance of the different steps of glycosylation for the activity and secretion of lipoprotein lipase in rat preadipocytes studied with monensin and tunicamycin

Lipoprotein lipase synthesized by cultured rat preadipocytes is present in three compartments: an intracellular, a surface-related 3-min heparin-releasable, and that secreted into the culture medium. 30 min after addition of 6 microM monensin, the lipoprotein lipase activity in the heparin-releasable compartment starts to decrease; by 4 h of monensin treatment the lipoprotein lipase activity in the heparin-releasable pool and in the culture medium is about 10% of that found in control dishes. The intracellular activity, which had been identified as lipoprotein lipase by an antiserum to lipoprotein lipase, increases slowly and doubles by 24 h. However, since the cellular compartment accounts for 10-25% of total activity, this increase does not account for the missing enzyme activity. To determine whether this enzyme molecule is synthesized but is not active, incorporation of labeled leucine, mannose and galactose into immunoadsorbable lipoprotein lipase was studied in control, monensin- or tunicamycin-treated cells. Addition of tunicamycin (5 micrograms/ml) for 24 h caused a 30-50% reduction in immunoadsorbable lipoprotein lipase, but the enzyme activity was reduced by 90%. On the other hand, 4 h monensin treatment reduced both incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase and heparin-releasable and medium lipoprotein lipase activity by 57 to 77%. The immunoadsorbable lipoprotein lipase in the intracellular compartment has a [14C]mannose to [3H]galactose ratio of 0.15 and this ratio increased 6-fold in monensin-treated cells. The intracellular lipoprotein lipase in monensin-treated cells had the same affinity for both the native and synthetic substrate as the lipoprotein lipase in control cells, yet its spontaneous secretion into the culture medium and its release by 3 min heparin treatment was markedly decreased. The present results indicate that: the presence of asparagine-linked oligosaccharide (formation of which is inhibited by tunicamycin) is mandatory for the expression of lipoprotein lipase activity; lipoprotein lipase is active also in a high mannose form; and terminal glycosylation and oligosaccharide processing, which is inhibited by monensin, may be important for the appearance of heparin-releasable lipoprotein lipase and secretion of lipoprotein lipase into the medium.     

Performance ofin vitro propagatedAlnus glutinosa (L.) Gaertn. clones inoculated withFrankiae

Summary ThreeAlnus glutinosa (L.) Gaertn. clones, obtained byin vitro propagation techniques, were inoculated with four strains ofFrankia. The ability of these clones to nodulate and fix nitrogen was previously reported; this study deals with the performance of 12 different combinations of pairs of symbionts.Shoot fresh weight, shoot height and collar diameter were measured 60 and 82 days after inoculation. Shoot fresh weight seems to be more sensitive and reliable than the other parameters. Nitrogenase activity, measured by the acetylene reduction assay, was assayed 78 days after inoculation and was consistent with the biomass measurements.Better growth was observed when type N strains were used. Significant growth differences were observed between clones AG-2 and AG-8 on the one hand and clone AG-4 on the other. Thus, the use of genetically defined host plants and microsymbionts permitted the demonstration of significant performance variation even among cloned plants from the same provenance (AG-4 and AG-8).The duration of the experiment influenced the results with differences becoming less significant with time. This might be caused by an external limiting factor such as the pot size, competition for light,etc. But it could also be indicative of differences in nodulation speed among the treatments.