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.     

Production of an antibody to mouse salivary androgen binding protein (ABP) and its use in identifying a prostate protein produced by a gene distinct from Abp

We previously demonstrated polymorphism of a mouse salivary protein which, because of its ability to bind androgen, we designated androgen binding protein (ABP) and its structural gene, Androgen binding protein (Abp). This report describes the purification of salivary ABP and presents the amino acid composition of its subunits. Using an antibody raised against the purified protein, we demonstrated the presence of cross-reactive material (CRM) in mouse parotid, submaxillary, sublingual, and prostate glands by double immunodiffusion. Immunohistochemical detection of proteins on electroblots of polyacrylamide electrophoresis and isoelectric focusing gels, however shows that the prostate CRM is a protein with electrophoretic and isoelectric focusing behavior distinct from that of salivary ABP isoproteins. Because the DBA/2J strain has a variant of salivary ABP, that strain was analyzed to determine if a prostate ABP-CRM variant was present. The approach was hampered by an inability to detect CRM in the prostates of DBA/2J mice. Prostate CRM was detected, however, in some progeny from repeated backcrosses of DBA/2J X C3H/St hybrids to the C3H/St and DBA/2J parental strains. The prostate CRM detected in samples from animals heterozygous for salivary Abp appears to be identical to C3H/St prostate CRM, suggesting that the gene controlling prostate ABP-CRM is related to, but distinct from, Abp. The reason for reduced or absent CRM in the prostates of DBA/2J males is unknown but this finding suggests that there are strain-related differences in the expression of this prostate protein.     

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.     

The amino acid sequence of the alpha subunit of mouse salivary androgen-binding protein (ABP), with a comparison to the partial sequence of the beta subunit and to other ligand-binding proteins

It has been suggested that three distinct genes,Abpa, Abpb, andAbpg, determine the three subunits of mouse salivary androgen-binding protein (ABP) (Dlouhy, S. R.,et al., Genetics 115, 535, 1987). We report the putative amino acid sequence of the subunit common to all forms of ABP, the Alpha subunit, and the partial amino acid sequence of the Beta subunit. These sequences have little in common, supporting the notion of at least two distinct genes coding for the subunits of the most common form of salivary ABP, the A:B dimer. A search of GenBank showed that these sequences have not been reported previously. The Beta subunit shows significant homology with helospectin, a member of the glucagon superfamily, but not enough homology to assign it to the family. No homology exists between ABP subunits and members of the ligand-binding carrier family of proteins nor does ABP show homology with other androgen-binding proteins. Particularly interesting is the observation that there is no relationship to rat prostatic steroid binding protein (PBP), given the similarities in protein tertiary structure, the numbers of subunits and their genes, and the earlier observation of ABP cross-reactive material in mouse prostate.Partial support for this work was provided by a PHS AREA award and by the Butler Academic Grants program. Both sources of support are greatly appreciated.A portion of this work constituted partial fulfillment of the honors thesis requirement for Butler University.     

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.     

Inheritance and biochemical analysis of four electrophoretic variants of β-conglycinin from soybean

Summary Three genes which code for variant -conglycinin subunits were identified. Alleles Cgy ₁ ^S and Cgy ₂ ^S were codominant with Cgy ₁ and Cgy ₂ and produced and subunits, respectively, with reduced electrophoretic mobility. Allele Cgy ₃ ^D increased the mobility of at least one polypeptide in the subunit family and exhibited incomplete dominance. Gene loci Cgy ₂/Cgy ₂ ^S and Cgy ₃ ^D /cgy ₃ ^D were linked, whereas Cgy ₁/Cgy ₁ ^S / cgy ₁ segregated independently of the others. Techniques developed for purification of normal -conglycinin subunits were effective in purifying the altered subunits. Deglycosylated variant proteins from seeds containing the alleles Cgy ₁ ^S , Cgy ₂ ^S , or Cgy ₃ ^D also has altered mobility relative to deglycosylated normal proteins. Therefore, the altered subunits contained changes in their amino acid sequences rather than in their carbohydrate moieties. This interpretation is consistent with the observed codominant or incompletely dominant mode of inheritance for these alleles and suggests that each contains an altered nucleotide sequence in the structural gene. A fourth variant, which exhibited doublet and a electrophoretic bands, was inherited in a recessive fashion. Deglycosylated subunit proteins from this variant were identical in electrophoretic mobility to those of the deglycosylated normal protein. This suggests that the doublet phenotype resulted from an alteration in the carbohydrate moiety of these subunits. The gene or genes which condition this variant presumably are required for normal post-translational modification of the subunit carbohydrates and as such may be useful for investigating these events.Cooperative research of USDA-ARS and the Indiana Agric. Exp. Stn., Purdue Univ., West Lafayette, IN 47907, USA. Indiana Agric. Exp. Stn. Journal Article 10,323. Financial support from the American Soybean Research Foundation is gratefully acknowledged     

Bacteriophage lambda FII gene protein: role in head assembly

The in vitro completion of bacteriophage lambda F_II^? heads to form phage can be used as an assay for the λ F_II gene protein. F_II protein activity is released from highly purified phage particles or phage heads by treatment with heat or denaturing agents. F_II protein was purified from isolated phage particles and from an extract of E^? infected cells in which it is not bound to any large structures. No differences in molecular weight (11,500), isoelectric point (4.75), electrophoretic mobility, or purification properties could be demonstrated between the F_II proteins from the two sources. Thus the polypeptide does not seem to be modified during assembly.Phage φ80 is closely related to λ. φ80 heads will join to φ80 tails in vitro but will not join to λ tails, though λ heads will join to either type of tail. Mixing experiments between F_II^? heads, tails, and F_II protein from λ or φ80 show that the specificity of head-tail joining is correlated with the source of the F_II protein and not with the source of the other head proteins. Thus, F_II protein is apparently responsible for this specificity of head-tail joining.     

Human partoid size polymorphism (Ps): Characterization of two allelic products,Ps 1 and 2, by limited proteolysis

The allelically determined human salivary proteins, Ps 1 and 2, were purified on sodium dodecyl sulfate gels, eluted, and compared by limited proteolysis with Streptomyces griseus protease VI, Bacillus subtilis protease VII, and Staphylococcus aureus protease V8. Prior dansylation of the Ps isoproteins facilitated visualization of the peptides. Digestion patterns indicate considerable homology between the Ps isoproteins and support the conclusion [Azen, A. E., and Denniston, C. (1980). Biochem. Genet. 18:483] that there is an actual molecular weight difference between them. Further, the results suggest that this difference owes to an extension of the Ps 2 chain at one of its ends.This study was supported in part by PHS Research Grant 1 RO1 DE05684. PAG was supported by NRSA Training Grant 5 T32 DE07043. RCK was supported by PHS Career Development Award 1 K04 AM00284.     

Molecular structure of rare but geographically widespread sn-glycerol-3-phosphate dehydrogenase ‘ultra-fast’ electrophoretic alleles in Drosophila melanogaster

Six naturally occurring but rare alleles of sn-glycerol-3-phosphate dehydrogenase (Gpdh) in Drosophila melanogaster have been investigated in this study. They all belong to a class of Gpdh ^UF (ultra-fast) alleles, because their electrophoretic mobilities are faster than that of the Gpdh ^F (fast) allele. The Gpdh ^UF variants are widespread, and have been reported from five continents. DNA sequence analysis has shown that the change in electrophoretic mobility was in each allele caused by a single amino acid residue substitution in the encoded protein. In the Xiamen ^UF allele it is a substitution of lysine (AAA) to asparagine (AAT) in exon 1 (residue 3). An asparagine (AAT) to aspartate (GAT) change was found in exon 6 (residue 336) in the Iowa ^UF and Netherlands ^UF alleles. The mobility of the Raleigh ^UF allele was altered by a valine (GTG) to glutamate (GAG) substitution in exon 3 (residue 76). Two mutations were detected in the Brazzaville ^UF allele: a lysine (AAG) to methionine (ATG) substitution in exon 2 (residue 68) is responsible for the ultra-fast phenotype of this variant, while a tyrosine (TAT) to phenylalanine (TTT) substitution in exon 4 (residue 244) is not expected to alter the electrophoretic mobility of the encoded protein. These results indicate that the Gpdh ^UF alleles originate from different mutational events, and only two of them — Iowa ^UF and Netherlands ^UF — might share a common ancestry. The GPDH activity of the Iowa ^UF allele is intermediate between those of the Gpdh ^S and Gpdh ^F control stocks. The other Gpdh ^UF variants have lower activities than the controls: Xiamen ^UF -83%, Raleigh ^UF -80% and Brazzaville ^UF -73% of the Gpdh ^F control.     

The structural gene for the ribosomal protein S18 in Escherichia coli. II. Chemical studies on the protein S18 having an altered electrophoretic mobility

A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region.     

The distribution of Gc subtypes among the Mongoloid populations

The polymorphism of Gc (group-specific components) has been investigated for a series of 3,160 individual samples from 11 Mongoloid populations in Asia and North and South America by isoelectric focusing on polyacrylamide gels. The samples fall into six Gc phenotypes which can be explained by the three common alleles, Gc^1F, Gc^1S, and Gc², together with several variant phenotypes explained as the heterozygotes for the three common alleles. The distribution of Gc^1F suballele appears to be considerably different from population to population among Mongoloids, ranging from 0.105 (Machiguenga Indans, Peru) to 0.609 (Kadazan, Borneo). A clear geographic cline from Southeast Asia into South America in Gc^1F allele was observed in the populations. In general, Gc^1F allele frequencies are lower in European populations and higher in African populations. The range of variability in the Gc^1F values observed among the Asiatic populations is between the Africans and the Europeans.     

Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints

We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone ¹⁵N T ₁/T ₂ data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T ₁/T ₂ ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.     

Evidence for duplication of the human salivary amylase gene

Summary Isoelectric focusing of human parotid saliva reveals different -amylase patterns reflecting qualitative and quantitative variations. A puzzling pattern, which shows three different amylase gene products, was found in four individuals. Based on this observation a model is presented in which the salivary amylase gene is duplicated. Family studies show that the AMY1 ^* A2 gene forms a haplotype with the normal gene, AMY1 ^* A1, whereas the AMY1 ^* A3 gene still exists in a single form. The absence of homozygote 2-2 in offspring of 1-2x1-2 marriages and in population material, and the fact that the variant protein makes up about only 20–30% of the total amylase protein in heterozygotes can be considered as additional evidence supporting the hypothesis. The possibility that cis-acting regulatory variants are involved in the patterns with quantitative variation is discussed.     

Genetic studies of murine catalase: Regulation of multiple molecular forms of kidney catalase

Starch gel electrophoresis of kidney catalase in inbred strains C3H and C57BL/6, their F₁ hybrid, and first and second backcross generations demonstrated that single-component (type A) v. multiple-component (type B) electrophoretic patterns are controlled by a single locus. The type A electrophoretic pattern is dominant. Twenty-five inbred strains of mice were classified according to their kidney catalase electrophoretic pattern. The data indicate that the segregating genetic factor determines a specific substance in the type A kidney which affects the electrophoretic mobility of catalase. A comparison of the F₁ hybrid enzyme with a 1:1 mixture of C3H and C57BL/6 enzyme showed that the alteration of electrophoretic mobility is the result of posttranslational modification of the catalase molecule. An association of kidney catalase electrophoretic pattern and the H-2 ^k haplotype indicates that the locus controlling the electrophoretic pattern is most likely located on chromosome 17 in close proximity to the H-2 complex.     

Inheritance of two endosperm protein loci in rice (Oryza sativa L.)

Summary Previous studies indicated two types of phenotypic protein markers as two minor bands of SDS-PAGE for rice storage protein. A variant derived from a Pakistani variety, Dular, was found to show a mobility variant with Band 11, a relatively faster-moving band as compared to Band 10, while most of the other cultivated rices exhibited Band 10 at a molecular weight of around 100–110 K. Band 11 was also observed in several wild rice species. How this variant occurred is not known. Another marker is characterized by the presence of either Band 56 (slower-migrating band) or Band 57 (faster-migrating band) in most cultivars at a molecular weight of about 28–27 K. Most indica varieties developed in Taiwan have Band 57 and japonica varieties have Band 56. Genetic analysis of F₁, F₂ and F₃ seeds from interstrain crosses indicated that Band 10 versus Band 11 and Band 56 versus Band 57 are due to codominant alleles at two loci. Tests of independent inheritance between these two loci (Band 10/11 versus Band 56/57) indicated that there is no linkage between them. Both of these two protein loci encode for endosperm proteins and mostly belong to the minor polypeptide subunits of the glutelin fraction of rice seed proteins. Studies on reciprocal crosses indicate dosage effects as exhibited in band patterns. Variations in band intensity were frequently observed when the maternal genotype was different.     

A new variant of human erythrocyte G6PD occurring at a high frequency amongst the population of two villages in The Gambia,West Africa

Summary During the course of a large survey of red cell G6PD genotypes in The Gambia, a slow electrophoretic variant with reduced enzyme activity was found to occur at a high frequency. This variant, G6PD Gambia, was found in the following genotypic combinations: males; G6PD^Gam, females; G6PD^A+/Gam, G6PD^B+/Gam, and G6PD^A-/Gam. From the electrophoretic mobility and kinetic characteristics it was concluded that G6PD Gambia was a hitherto unreported variant of G6PD. The frequency of the G6PD^Gam gene amongst the 1109 individuals examined was 0.024.     

An acid phosphatase locus expressed in mouse kidney (Apk) and its genetic location on chromosome 10

A genetic locus controlling the electrophoretic mobility of an acid phosphatase in mouse kidney is described. This locus, called acid phosphatase-kidney (Apk), is not expressed in erythrocytes, liver, spleen, heart, lung, brain, skeletal muscle, stomach, or testes. The product of Apk hydrolyzes the substrate naphthol AS-MX phosphoric acid but is not active on a-naphthylphosphate or 4-methylumbelliferylphosphate. It is not inactivated by 50 C for 1 hr, nor is its electrophoretic mobility altered by incubation with neuraminidase. The locus is invariant among 31 inbred strains (Apk ^a), with a variant allele (Apk ^m) observed only in Mus musculus molossinus. Codominant expression was observed in F₁ hybrids of M. m. molossinus and inbred strains. Apk was mapped on Chr 10, near the neurological mutant waltzer (v).This work was supported by Contract NO1-ES42159 from the National Institute of Environmental Health Sciences and by Grant 1-476 from the National Foundation—March of Dimes. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

The mechanism of sex determination in Rumex acetosella

Summary Cytogenetic studies were made with particular emphasis on the sex-determining mechanism in Rumex acetosella (6 x = 42) and its hybrids (F ₁, F ₂, BC ₁ and BC ₂) with R. hastatulus (synthetic 4 x = 16 = 4 A +4 X = and 4 x = 18 = 4 A + 2 (X Y ₁ Y ₂) = ). Rumex acetosella was almost strictly dioecious with 5050 male and female. Breeding tests revealed that the males were heterogametic. The longest chromosomes (S), usually two, are the sex chromosomes of this hexaploid species. The S chromosomes are homomorphic in both male and female. The sex chromosome: autosome ratios, and the strong epistatic male effect of the S ^M chromosome in the polyploid dioecious species and in the hybrids, are evidence of an X/Y Melandrium type sex-determining mechanism controlled by a single pair of homomorphic sex chromosomes. Thus, the sex chromosome formula of the males was S ^F S ^M and that of females was S ^F S ^F. The present approach is a new method for resolving the sex-determining mechanism in a dioecious species.