Immunological relationships and post-translational modifications of human salivary amylase (Amy1) and pancreatic amylase (Amy2) isozymes

Electrophoretic phenotypes of human salivary amylase (Amy₁) and pancreatic amylase (Amy₂) consist of complex isozyme patterns which may result from post-translational modifications of the primary products of the amylase loci. Biochemical separation of the two molecular weight families of salivary amylase and development of a new electrophoretic system have allowed the identification of complete isozyme patterns corresponding to variant alleles in Amy ₁ and Amy₂ heterozygotes. Further, immunological studies show no nonidentities among salivary isozymes and among pancreatic isozymes, which is to be expected if each series is derived from a single gene product. Both results support the hypothesis that the primary products of the amylase loci undergo post-translational modifications. Salivary and pancreatic amylase appear to be immunologically identical.This investigation was supported in part by PHS Research Grant GM-19178.Supported by PHS Training Grant DE 119.Supported by PHS Training Grant GM 1056.     

Sex-limited genetic variation in a mouse salivary protein

This report describes a gene which influences the electrophoretic mobility of a protein in the salivas of adult mice. Three categories of phenotype have been observed: the two single-banded types, F (Fast) and S (Slow), and the two-banded type, SF (Slow-Fast), with the two bands represented in varying proportions. All females, regardless of age or strain, and all males before puberty show only the F phenotype. Males of the BALB/c and C57BL/6J strains show the F phenotype throughout puberty and adult life, whereas males of the C3H/St and C57BL/KsJ strains show the SF phenotype in puberty and the S phenotype in adult life. We have designated this variation the sex-limited saliva pattern (Ssp). The results from genetic crosses indicate that the variation among the strains is determined by an autosomal locus, Ssp, with two alleles, Ssp ^S andSsp ^F ,where Ssp ^S is dominant to Ssp ^F .Testosterone treatment can accelerate the acquisition of the S type in males of the strains C3H/St and C57BL/KsJ and also induces that phenotype in C3H/St females and C57BL/6J males. Thus it appears that the observed strain-specific differences reflect a genetic variation in androgen levels and/or androgen sensitivity rather than variation in a structural gene.This study was supported in part by PHS Research Grant 5 RO1 AM21177 and by the Indiana University Human Genetics Center (PHS PO1 GM21054). The preliminary work was done during a 1-month visit by RCK to the Institute of Ecology and Genetics which was supported by the University of Aarhus. This is publication No. 80-18 from the Department of Medical Genetics. RCK was supported by PHS Career Development Award 1 KO4 AM00284. SRD was supported by PHS General Medical Training Grant T32 GM07468.     

Polymorphism of Ps (parotid size variant) and detection of a protein (PmS) related to the Pm (parotid middle band) system with genetic linkage of Ps and Pm to Gl,Db, and Pr genetic determinants

Genetic polymorphism of the Ps (parotid size variant) proteins found in saliva is determined by autosomal inheritance of two expressed and one unexpressed allele. This hypothesis is supported by studies in 43 families including 153 children. Gene frequencies determined for 150 randomly collected salivas from whites and 101 randomly collected salivas from blacks are as follows: for whites, Ps ¹=0.598, Ps ²=0.101, Ps ⁰=0.301; for blacks, Ps ¹=0.185, Ps ²=0.126, and Ps ⁰=0.689. The electrophoretic polymorphism is manifested by apparent differences in molecular weights between Ps proteins. The Ps proteins are glycosylated and have an approximate isoelectric point of pI 8.1 as determined by isoelectric focusing in gels. We have also found in saliva the presence of a protein (PmS) which shows strong positive correlations with the presence of the smaller sized Pm (PmF) salivary protein described by Ikemoto et al. (1977). This suggested that PmS is probably part of the Pm protein polymorphic system. For randomly collected salivas from whites, the gene frequencies are PmF+=0.15 (N=140) and PmS+=0.12 (N=150). For randomly collected salivas from blacks, the gene frequency is PmS+=0.24 (N=101). The gene frequency of PmF+ was not determined. Family studies support autosomal inheritance of PmF and PmS proteins. There is strong evidence for linkage of Pm to the proline-rich protein (PPP) region (11 families, lod score at =0.01 is 7.64) and of Ps to the PPP region (13 families, lod score at =0.01 is 11.50). Protein products of six linked loci (Pr, Pa, Db, Ps, Pm, and Gl), when tested against rabbit anti-Gl antiserum, show immunological reactions of partial or complete identity with each other by double diffusion analysis.This study was supported by a grant from the National Institutes of Dental Research (DEO 3658-14). Paper No. 2340 of the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706.     

Multiple gene action determining a mouse salivary protein phenotype: Identification of the structural gene for androgen binding protein (Abp)

A novel variation in electrophoretic phenotype is described for a mouse salivary androgen binding protein (Abp). Crosses show that the variation is inherited in an autosomal codominant manner and protein characterization studies show that the variant Abp differs in isoelectric point from the common form of the protein. Those observations suggest that the variation involves the structural gene for the mouse salivary Abp. The genetic studies also show that the electrophoretic mobility of the variant Abp can be influenced by the sex-limited saliva pattern (Ssp) gene. The Ssp ^S allele alters the electrophoretic mobility of Abp in males at puberty or in females which have received exogenous testosterone [Karn, R. C., Dlouhy, S. R., Springer, K. R., Hjorth, J. P., and Nielsen, J. T. (1982). Biochem Genet. 20:493]. This study shows that Abp and Ssp are distinct genes which are not closely linked and that Ssp ^S is trans active in F₁ (Abp ^a /Abp ^b , Ssp ^S /Ssp ^F ) males.SRD was supported in part by PHS General Medical Training Grant T32 GMO7468 and the Indiana University School of Medicine Research Program in Academic Medicine. RCK was supported in part by PHS Career Development Award 1 KO4 AMOO284.     

Phylogenetic, structure and expression analysis of ABC1Ps gene family in rice

The ABC1 protein family (ABC1P), a new family of putative kinases, widely existed in procaryote and eucaryote. A comprehensive genome-wide analysis was carried out in this study to find all ABC1Ps in rice (Oryza sativa subsp. japonica). We identified 15 ABC1P genes in rice. All the ABC1Ps contained an ABC1 domain of about 120 conserved amino acid residues and conserved kinase motifs-VAIK (VAVK, VAMK) and DFG (DEG). The phylogenetic analysis showed that all the ABC1Ps were grouped into three subgroups, and formed a total of 12 sister pairs. Conserved motifs analysed by MEME program indicated that almost all ABC1Ps contains motifs 1, 3, 7, 8 and 9. Predictably, the ABC1Ps were localized in mitochondria or chloroplasts, which implied that the ABC1Ps might be involved in energy metabolism in plants. RT-PCR assays demonstrated that all 15 ABC1P genes were active, and some of them were affected by abiotic stresses (NaCl, high temperature, methyl viologen, abscisic acid and cadmium).     

Structural homology between Drosophila melanogaster and Escherichia coli acidic ribosomal proteins

Antibodies raised against Drosophila melanogaster ribosomal proteins (r-proteins) were used to examine possible structural relationships between eukaryotic and prokaryotic r-proteins. The antisera were raised against either groups of r-proteins or individually purified r-proteins. Two antisera showed a cross-reaction with total Escherichia coli r-proteins in Ouchterlony double immunodiffusion assays: an antiserum against the D. melanogaster small subunit protein S14 (anti-S14) and an antiserum against a group of D. melanogaster r-proteins (anti-TP80). The specificity of the antisera and the identity of the homologous E. coli r-proteins were characterized by using immunooverlay and immunoblot assays. These assays indicated that anti-S14 was highly specific for protein S14 and anti-TP80 was a multispecific serum that recognized several of the D. melanogaster ribosomal proteins. The E. coli protein homologous to D. melanogaster protein S14 was identified as E. coli protein S6. By adsorption of the anti-TP80 serum, we determined that D. melanogaster protein 7/8 is homologous to the acidic E. coli protein L7/L12. D. melanogaster acidic protein 13 was also shown to be immunologically related to D. melanogaster protein 7/8.This research was supported by National Institutes of Health Grant GM23410 awarded to WYC. LMS was the recipient of a predoctoral fellowship from Molecular, Cellular, and Developmental Biology Training Grant PHS T32 CM07227. We are very grateful to Dr. Anthony Mahowald for providing us with embryos.     

Genetic analysis of alcohol dehydrogenase isozymes in pearl millet (Pennisetum typhoides)

Pearl millet (Pennisetum typhoides) produces three ADH isozymes, sets I, II, and III, with set III being expressed only in anaerobically treated seeds or seedlings. Variant strains have been identified which produce ADH isozymes with altered electrophoretic mobilities for sets I and II but not for set III activity. Based on genetic analysis of these variants and on dissociation-reassociation experiments, we propose that the three ADH isozymes are dimers of subunits coded by two structural genes, Adh1 and Adh2, with set I being a homodimer specified by Adh1, set III a homodimer specified by Adh2, and set II a heterodimer formed between the products of Adh1 and Adh2.This work was supported by BRSG Grant RR 07080 awarded by the Biomedical Research Grant Program, Division of Research Grants, National Institutes of Health, to D. R. H., and by funds from the Margenroth Endowment to F. B.-B., who is a PHS Research Service Award Trainee in Genetics.     

Segregation distorter in D. melanogaster males: An effect of female genotype on recovery

Summary Segregation Distorter (SD) is a gene affecting sperm recovery in Drosophila melanogaster. In a cross SD/SD ⁺ x SD⁺, the proportion of SD/SD ⁺ zygotes recovered (k) is larger than the 0.50 expected. Previous investigations have shown that the relative recovery of SD- and SD ⁺-bearing sperm is determined at or before the meiotic divisions. Evidence is presented here that k is influenced by the genotype of the female parent. It is suggested that the SD gene product causes a difference in the enzymatic or structural complement of the SD- and SD ⁺-bearing sperm which largely determines their relative functionality. The effect of the female genotype on recovery is interpreted as an interaction between this physiological difference and the environment of the female reproductive tract.Adapted from a dissertation presented by the senior author in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported by PHS Training Grant No. GM 00337 and PHS Research Grant No. GM 12334 from the National Institute of General Medical Sciences.     

Genetic analysis of a meiotic mutant resulting in precocious sister-centromere separation in Drosophila melanogaster

Summary It is shown that mei-S332, a semidominant mutant in Drosophila melanogaster, has the following properties: 1) It maps at about position 95 on the right arm of chromosome 2. 2) Its primary result is the precocious division of sister centromeres, which leads to nondisjunction, mostly equational, and loss for all chromosomes in both sexes. 3) Chromosome pairs behave approximately independently. 4) Gamete types for each chromosome appeared in the approximate ratios of 0.20 nullo-gametes: 0.12 diplo-gametes: 0.68 regular gametes for experiments reported here. 5) Exchange is correlated with a lower probability of reductional nondisjunction, but is independent of the probability of equational nondisjunction. 6) Since the phenotype of mei-S332 is similar in the two sexes, at least part of the meiotic events and their control for the second division and perhaps also the first division is the same in the two sexes. 7) Mosaics, resulting from mitotic chromosome loss appear among progeny of mei-S332 parents.Adapted from a dissertation presented in partial fulfillment of the degree of Doctor of Philosophy. The research was supported by a PHS Training Grant and PHS Grant RG-9965.     

Identification of a Proteolytic Bacterium,HW08, and Characterization of Its Extracellular Cold-Active Alkaline Metalloprotease Ps5

A psychrophilic protease-producing bacterium, HW08, was isolated from sediment of the Yellow Sea in eastern China. On the basis of 16S rDNA sequence analysis and physiological properties, the isolate was identified as Pseudomonas lundensis. The secreted protease, named Ps5, was purified from the culture supernatant as a monomer with an apparent molecular mass of 46 kDa on SDS–PAGE. As a metalloprotease (inhibited by EDTA), the enzyme showed maximum activity at 30 °C at pH 10.4. It had no activity loss exposed at 4 °C for 60 d or under repeated freezing and thawing. Broad temperature (25–40 °C) and pH (7.0–11.0) stability was observed in the presence of 5 mm Ca²⁺. Furthermore, the enzyme was resistant to detergent additives such as non-ionic surfactants and bleaches. It showed considerable potential for industry that requires alkaline-protease.     

In vitro translation of maize ADH: Evidence for the anaerobic induction of mRNA

Total cellular RNA from anaerobically stressed maize seedling roots was used to stimulate in vitro translation of authentic maize alcohol dehydrogenase (ADH) in a rabbit reticulocyte lysate system. Total products from such reactions were displayed on NEPHGE-SDS two-dimensional gels and the Adhl-specific translation products were identified by using RNA from sib seedlings segregating for Adhl charge and size variants. The application of a rapid RNA isolation procedure allowed the efficient isolation of biologically active RNAs from small amounts of seedling material. Maize ADHs translated in vitro are identical in size to in vivo ADH. Further, no ADH was detected in the products of an in vitro translation reaction stimulated by total RNA from aerobically grown seedlings. This suggests that induction of ADH protein by anaerobic stress is accomplished by production of Adh mRNA rather than activation of sequestered mRNA. The mRNAs for maize ADH1 and ADH2 are among a small class of mRNAs induced during anaerobiosis.Research was supported by NSF Grant PCM 76-11009. M.D.B. is supported by National Institute of Health Grant PHS 5 T32 GM07227-04. R.J.F. is a Predoctoral Trainee in Genetics supported by National Institute of Health Training Grants 82 and 7757 from the National Institute of General Medical Sciences.     

Sex-limited effects of the expression of the db gene in mice during puberty

The autosomal recessive gene diabetes (db) produces a condition similar to human insulin-dependent diabetes mellitus in certain strains of inbred mice. In this investigation, the effects of expression of the db gene on the development of the submandibular glands, electrophoretic protein patterns in salivas, fasting blood glucose levels, and glycosylated hemoglobin levels were evaluated in mice undergoing puberty. Three sex-limited effects of the db gene were observed in diabetic male mice: (1) a compromise of the development of the specialized submandibular glands with the extensive tubular portion normally found in males, (2) failure to develop a salivary protein pattern unique to male mice, and (3) attainment of higher levels of fasting blood glucose than found in female diabetic mice. Since it has been documented that homozygous mice fail to develop functional gonads, apparently due to insufficient production of gonadotropin, it is likely that the compromised development of the specialized submandibular glands, and, consequently, the male salivary protein pattern, is a result of decreased testosterone production. Experiments in which diabetic mice were treated with testosterone support that conclusion, since testosterone caused transformation of the salivary protein pattern to one identical with that of normal male littermate controls and increased the tubular portion of the submandibular glands.This study was supported in part by Public Health Service Research Grant 5 R01 AM21177 and by the Indiana University Human Genetics Center (PHS P01 GM21054). The author was supported by Public Health Service Career Development Award 1 K04 AM00284. This is Publication No. 79-18 from the Indiana University Human Genetics Center.     

Social interactions of juvenile female bonnet monkeys,Macaca radiata

This study reports observations on juvenile female bonnet macaques (Macaca radiata) in a field cage environment. Interaction and proximity data collected on different age classes of juvenile females indicate distinct changes in social behavior from the time a female is weaned until her first pregnancy. Behavioral development includes an increase in solitary behavior, an early disinterest in younger animals, and a surprisingly low interaction rate with relatives. Important aspects of ontogeny are lost in analyses that treat all juvenile females as a single age-class. This research was supported partially by US PHS Grant No. RR00169.     

Linkage of the alcohol dehydrogenase structural genes in pearl millet (Pennisetum typhoides)

Pearl millet produces three ADH isozymes, Sets I, II, and III. Naturally occurring ADH electrophoretic variants affecting Sets I and II isozymes but not III have been previously described. Analysis of such variants led to the identification of the Adh1 structural gene. The existence of a second Adh structural gene was inferred from dissociation-reassociation studies of Set II. In the present report, a naturally occurring variant affecting the electrophoretic mobility of Sets III and II but not Set I is described. Analysis of this variant confirms the existence of a second structural gene, Adh2. Crosses utilizing this Adh2 marker reveal a dissimilarity with maize and other plants such as sunflower and narrow-leafed lupins. Adh1 and Adh2 of pearl millet do not segregate independently; indeed, no recombinants have been observed. This is the first major difference encountered in an otherwise remarkably similar genetic and environmental control of the ADH isozymes in maize and millet. The organization of the Adh genes of pearl millet may reflect a more primitive arrangement than that of maize.This work was supported by a PHS National Research Service Award Training Grant in Genetics to the Biology Department of the University of Oregon.     

Studies of esterase 6 in Drosophila melanogaster. I. The genetics of a posttranslational modification

A locus has been found, an allele of which causes a modification of some allozymes of the enzyme esterase 6 in Drosophila melanogaster. There are two alleles of this locus, one of which is dominant to the other and results in increased electrophoretic mobility of affected allozymes. The locus responsible has been mapped to 3-56.7 on the standard genetic map (Est-6 is at 3-36.8). Of 13 other enzyme systems analyzed, only leucine aminopeptidase is affected by the modifier locus. Neuraminidase incubations of homogenates altered the electrophoretic mobility of esterase 6 allozymes, but the mobility differences found are not large enough to conclude that esterase 6 is sialylated.This work was supported by NIH Grant No. GM23706 and PHS Grant SO7RR7031 to Rollin C. Richmond and by NIH Genetics Training Grant No. 82 to Indiana University.     

Isozyme variability in species of the genus Drosophila. VII. Genotype-environment relationships in populations of D. melanogaster from the eastern United States

Collections of D. melanogaster were analyzed for genetic variation at nine enzyme loci. Allelic frequencies were determined, and comparisons between observed phenotypic proportions and those expected under equilibrium conditions were made. A significant tendency toward homozygote excess was noted. Relationships between patterns of genetic variability and between patterns of genetic and environmental variability were examined by the method of principal components and correlation analysis. Several of the loci showed a clinal pattern in gene frequencies, and overall there was an appreciable amount of genetic-genetic and genetic-environmental association.This work was supported by PHS Research Grant No. GM 11546 and AEC Contract No. AT-(40-1)-3980. Paper No. 4026 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.     

Comparative biochemistry of murine arylsulfatase B

Arylsulfatase B was purified 4500-fold from liver and kidney of C57BL/6J mice. Hepatic and renal arysulfatase B are apparently determined by a single structural locus; however, posttranslational modification introduces inter- and intratissue microheterogeneity. Partially purified enzyme from C57BL/6J, A/J, C3H/HeJ, and SWR/J mice has similar catalytic properties. The 4500-fold-purified arylsulfatase B from SWR/J and C3H/HeJ mice was more thermostable than that from C57BL/6J and A/J mice, strongly suggesting that the thermostability difference reflects an alteration of the primary structure of the enzyme. Thermal stability of arylsulfatase B was pH dependent and markedly influenced by buffer anion. Variation of thermostability did not appear accountable for the observed activity variation among these strains; however, this possibility cannot be rigorously excluded by presently available data. Thirty-five murine strains were found to possess the As-1 ^a allele (thermostable enzyme), while As-1 ^b was largely restricted to A and C57 strains.This research was supported by PHS Biomedical Sciences Research Support Grant RR-07030.     

Exopolysaccharides of the plant pathogens Pseudomonas corrugata and Ps. flavescens and the saprophyte Ps. chlororaphis

The rRNA-DNA homology group I pseudomonads Pseudomonas asplenii, Ps. corrugata, Ps. flavescens (plant pathogens), Ps. alcaligenes, Ps. pseudoalcaligenes subsp. pseudoalcaligenes (opportunistic human pathogens), Ps. aureofaciens and Ps. chlororaphis (saprophytes) were examined for their ability to produce exopolysaccharides (EPSs) when cultured on various solid and liquid complex media with glucose, glycerol or gluconate as primary sources of carbon. All three strains (388, 717 and ATCC 29736) of Ps. corrugata produced alginate, a polyuronan. An EPS composed of glucose, fucose, mannose and an unidentified uronic acid substituted with lactic acid was produced by one (B62) of two strains of Ps. flavescens. Of four strains of Ps. chlororaphis tested, only strain NRRL B-2075 produced EPS. The extracellular material purified by anion-exchange chromatography appeared to be a mixture of alginate plus an acidic hexosamine-containing polymer(s). Production of EPS by the other pseudomonads was not supported by any of the media tested.     

Inhibitory effects of organic sulfur compounds on Histoplasma capsulatum

Summary Eight classes of organic sulfur compounds, comprising 42 substances, were tested in vitro for activity against the yeast phase ofHistoplasma capsulatum Darling. Activity was found in the classes of thiols, thio acids, disulfides, and thiolsulfonates. The classes of sulfinates, sulfenamides, penicillamine analogs, and reaction products of thiols with aldehydes or ketones were unpromising. Tentative conclusions are drawn of structure-activity relations within classes as a guide to coupling of classes to form disulfides.This investigation was supported by Biomedical Science Support Grant, National Institutes of Health Grant FR-07089-02 to Vanderbilt University (Ilda McVeigh) and by PHS Research Grant AM-11685 from the National Institute of Arthritis and Metabolic Diseases (Lamar Field).     

PRBI gene variants coding for length and null polymorphisms among human salivary Ps, PmF, PmS, and Pe proline-rich proteins (PRPs).

Six closely linked PRP (proline-rich protein) genes code for salivary PRPs that show frequent length and null polymorphisms. We report assignment of Ps proteins to the PRB1 gene, the derived primary structures of Ps 1 and Ps 2 proteins, and the molecular basis for some null alleles among PRB1-coded PRPs (Ps, PmF, PmS, and Pe). The derived primary structures of Ps 1 and Ps 2 proteins were determined by sequencing exon 3 of the different-length PRB1M (medium) and PRB1L (large) copies from subject C.J. with the Ps 1-2 phenotype. The PRB1L copy (coding for Ps 2) contained three additional tandem repeats within the Ps coding region, and the different-length Ps 1 and Ps 2 proteins can be explained on this basis. The molecular basis for the Ps 0 and the Pe- phenotypes was determined in another individual (M.V.O., a PRB2/1 fusion-gene heterozygote) with a single PRB1L copy. A premature stop mutation (CGA [Arg]-->TGA [stop]) occurred at residue 61 in the Ps-coding region. The identical mutation was found in the PRB1L and PRB1/2S (small) copies of a second individual (E.A.) with reduced Pe protein and the Ps 0 phenotype. This individual is a PRB1/2 fusion-gene heterozygote (Azen et al. 1992) with probably three mutated PRB1 copies (PRB1L-PRB1L-PRB1/2S). DNA sequences of the postulated crossover region of the PRB1/2S fusion-gene copy supported the postulated crossover. The PmF- and PmS- phenotypes in the three subjects were due to both the stop mutation and the lack of suitable proteolytic cleavage sites in the PRB1-coded precursor proteins.