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.     

Congenic strain analysis reveals genes that are rapidly evolving components of a prezygotic isolation mechanism mediating incipient reinforcement

Two decades ago, we developed a congenic strain of Mus musculus, called b-congenic, by replacing the androgen-binding protein Abpa27(a) allele in the C3H/HeJ genome with the Abpa27(b) allele from DBA/2J. We and other researchers used this b-congenic strain and its C3H counterpart, the a-congenic strain, to test the hypothesis that, given the choice between signals from two strains with different a27 alleles on the same genetic background, test subjects would prefer the homosubspecific one. It was our purpose in undertaking this study to characterize the segment transferred from DBA to the C3H background in producing the b-congenic strain on which a role for ABPA27 in behavior has been predicated. We determined the size of the chromosome 7 segment transferred from DBA and the genes it contains that might influence preference. We found that the "functional" DBA segment is about 1% the size of the mouse haploid genome and contains at least 29 genes expressed in salivary glands, however, only three of these encode proteins identified in the mouse salivary proteome. At least two of the three genes Abpa27, Abpbg26 and Abpbg27 encoding the subunits of androgen-binding protein ABP dimers evolved under positive selection and the third one may have also. In the sense that they are subunits of the same two functional entities, the ABP dimers, we propose that their evolutionary histories might not be independent of each other.     

Biochemical and immunological characterization of genetic variants of phosphoglucose isomerase from mouse

Two electrophoretic variants of phosphoglucose isomerase (PGI) were purified from whole body extracts of DBA/2J and C57BL/6J mice by a substrate-affinity elution from an 8-(6-aminohexyl) amino-ATP-Sepharose column followed by preparative isoelectric focusing. Both PGI variants were shown to be dimers of the same molecular weight, sedimentation coefficient, and K _m for fructose-6-phosphate. The isoelectric points were found to be 8.4 and 8.7 for variants from DBA/2J and C57BL/6J mice, respectively. Differential thermal stability was observed for the two variants in 0.1 m tris-HCl buffer, pH 8.0, at 54 C; the half-lives of the purified PGI from DBA/2J and C57BL/6J mice were shown to be 3.4 and 1.8 min, respectively, under those conditions. Similar differences were observed for the enzyme variants in the crude homogenates. Antisera against PGI from DBA/2J mice were raised in rabbits. The variants from DBA/2J and C57BL/6J mice showed no significant differences in their respective inactivation curves by the antisera. Results of amino acid composition analyses and peptide mappings of the two PGI variants indicate that the genetic variation of this enzyme might result from a single charged amino acid substitution.D. J. C. is a National Institutes of Health Visiting Fellow.     

Biochemical characterization of phosphoglucose isomerase and genetic variants from mouse and Drosophila melanogaster

Summary A simple and unique procedure was developed to purify phosphoglucose isomerase variants from the whole mouse body extracts and Drosophila homogenate. It involved the use of an 8-(6-aminohexyl)-amino-ATP-Sepharose column followed by a preparative isoelectric focusing. In each case, the enzyme in the homogenate was adsorbed by ionic interaction on the ATP-Sepharose column. Substantial purification was achieved by the affinity elution with the substrate-glucose-6-phosphate. Mouse and Drosophila phosphoglucose isomerase as well as the corresponding variants were shown to be dimers of similar molecular weight and to exhibit similar kinetic properties. The isoelectric points for the variants from DBA/2J and C57BL/6J mice were determined to be 8.4 and 8.7 respectively, while they were 6.8 and 6.3 respectively for Drosophila and 4/4 variants. Differential thermal stability was observed for the two mouse variants but not for the Drosophila ones. Amino acid composition analysis was performed for both mouse and Drosophila enzymes. Rabbit antisera for mouse (DBA/2J) and Drosophila (2/2) enzymes were raised. Within each species, complete immunological identity was observed between the variants. The antisera were used to characterize the null mutants of phosphoglucose isomerase identified in the mouse and Drosophila populations. By rocket immunoelectrophoresis, the null allele of the naturally occurring heterozygous null variant of Drosophila was shown to express no cross-reacting materials (CRM).     

Androgen repression of the production of a 29-kilodalton protein and its mRNA in the rat ventral prostate

The regression of the ventral prostate, after a rat is deprived of androgens by castration, is accompanied by a marked decrease in the prostate's ability to synthesize RNA and major proteins. Surprisingly, in vitro translation of prostate RNA, isolated from rats 2 days after castration, detects four proteins with Mr of approximately 29,000, 37,000, 46,000, and 49,000 whose message levels increased 4- to 12-fold compared to results from normal rats. According to cDNA dot hybridization analysis, the increase after castration in the level of the 29-kDa protein-mRNA (per unit amount of DNA) was reversed within 6 h by androgen treatment of castrated rats. In contrast, the level of a mRNA in male rat liver, which hybridized to a cloned probe for the prostate 29-kDa protein-mRNA was reduced by castration and increased by androgen treatment. During an in vitro incubation, the ventral prostates of normal rats were much less efficient than the prostates of rats castrated 2 days earlier in synthesizing a 29-kDa protein. Despite the fact that androgenic manipulation of rats induced very rapid and significant changes in the production of the 29-kDa protein and in the level of its mRNA, the cellular level of this protein in the prostate, as determined by radioimmunoassay, was maintained at near normal values throughout the 2-week experimental period. Thus, the prostate appears to have a mechanism, based on androgen repression of certain genes, to maintain the cellular levels of the 29-kDa protein and possibly other structurally or functionally important proteins during both the periods of androgen-dependent growth and the castration-induced regression. The loss of such a regulatory mechanism may result in androgen-independent abnormal prostate growth.     

Clearance, metabolic fate and tissue distribution of an injected bolus of photoaffinity-labeled rat androgen binding protein

An injected bolus of tritiated photolabeled rat androgen binding protein (ABP) is cleared from the circulation in a biphasic manner. The rapid component of the clearance curve indicates a clearance half time (t1/2) of 4.24 +/- 0.20 h and the slow component indicates a t1/2 of 13.27 +/- 2.92 h. After injection, photolabeled ABP is rapidly degraded as determined by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. This method dissociates ABP into subunits of approximately 48,000 daltons. One-half hour after injection of photolabeled ABP, 90% of the radioactivity migrates as a 20,000- rather than as a 48,000-dalton moiety; at the 24-h point only 20,000- and 16,000-dalton species were detectable. Chromatography of plasma on Sephadex G-150 indicated that, under nondenaturing conditions, radioactivity was still associated with proteins having a molecular weight similar to the native ABP dimer. These results suggest that the dimer is substrate for the proteolytic processes affecting ABP. Small (congruent to 7000 dalton) fragments of ABP could be detected in urine by G-150 chromatography 2 h after injection. The control protein [3H] bovine serum albumin (BSA) was cleared from the blood with a time course identical to that of photolabeled ABP (t1/2's 4.69 +/- 0.09 h and 13.56 +/- 1.03 h). [3H] BSA was degraded to peptides that were similar in size to those formed from photolabeled ABP. The uptake of photolabeled ABP and [3H] BSA by potential target tissues (testis, epididymis and ventral prostate) was equivalent, suggesting that the uptake of ABP was a nonspecific phenomenon rather than a receptor-mediated event. The liver and kidneys contained more radioactivity that did the other tissues and there was a differential uptake and clearance of photolabeled ABP and [3H] BSA by these organs. Whether this difference is related to specific uptake processes or to differences in the size and/or structure of the proteins or other factors remains to be determined.     

Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions.     

Androgen Receptor Splice Variant AR3 Promotes Prostate Cancer via Modulating Expression of Autocrine/Paracrine Factors

Deregulation of androgen receptor (AR) splice variants has been implicated to play a role in prostate cancer development and progression. To understand their functions in prostate, we established a transgenic mouse model (AR3Tg) with targeted expression of the constitutively active and androgen-independent AR splice variant AR3 (a.k.a. AR-V7) in prostate epithelium. We found that overexpression of AR3 modulates expression of a number of tumor-promoting autocrine/paracrine growth factors (including Tgfβ2 and Igf1) and expands prostatic progenitor cell population, leading to development of prostatic intraepithelial neoplasia. In addition, we showed that some epithelial-mesenchymal transition-associated genes are up-regulated in AR3Tg prostates, suggesting that AR3 may antagonize AR activity and halt the differentiation process driven by AR and androgen. This notion is supported by our observations that the number of Ck5⁺/Ck8⁺ intermediate cells is increased in AR3Tg prostates after castration, and expression of AR3 transgene in these intermediate cells compromises prostate epithelium regeneration upon androgen replacement. Our results demonstrate that AR3 is a driver of prostate cancer, at least in part, through modulating multiple tumor-promoting autocrine/paracrine factors.     

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.     

Mouse senile amyloidosis. ASSAM amyloidosis in mice presents universally as a systemic age-associated amyloidosis.

Amyloid deposition in 11 inbred strains of mice (A/J, SJL/J, DDD, C57BL/6J, B10.BR, C57BL/10, B10A/SgSn, C3H/HeMs, B10A(5R), DBA/2 and C57BL/6Cr5/c) was studied using the peroxidase antiperoxidase (PAP) method and antisera against ASSAM and murine protein AA. Among the 170 mice examined, in 77 (45.3%) from the nine strains other than C3H/HeMs and DBA/2, there was evidence of spontaneous amyloid deposits in routine histological sections. Immunohistochemical studies using 54 mice with amyloid deposition, demonstrated ASSAM deposition in 45 mice (83.3%) in all nine strains, although the incidence and intensity of the deposition differed somewhat between strains. SJL/J and A/J had ASSAM deposits from the age of 8 months and the incidence increased with advancing age. In the other seven strains, ASSAM was first deposited at an older age than in the SJL/J and A/J strains. In A J, C57BL/6J, C57BL/10, B10.BR, B10A(5R) and C57BL/6Cr5/c, protein AA often coexisted with ASSAM. The distribution pattern of the ASSAM deposits was similar to that observed among the SAM strains. Thus, ASSAM is an ubiquitously distributed senile amyloid protein in the mouse. Determination of the molecular type of apoA-II, a serum precursor of ASSAM, among all 11 strains using the polymerase chain reaction (PCR) revealed the SAM-P/1 type apoA-II variant in SJL/J and A/J strains with a high susceptibility to ASSAM deposition. We concluded from this study that amino acid substitution in precursor apoA-II may be responsible for the early onset and severe amyloid deposition in the mouse.     

A sex-limited serum protein variant in the mouse: Hormonal control of phenotypic expression

The sex-limited protein (Slp) antigen of the mouse is first detected in the serum of strain DBA/2J males at 5–6 weeks of age and reaches full adult levels by 10 weeks. This antigen is normally absent in females. Immature DBA/2J males castrated at 3 1/2 weeks of age failed to develop Slp antigen, while DBA/2J females treated with testosterone propionate starting at 3 1/2 weeks developed normal adult male levels of Slp antigen. Similar hormone-influenced effects were demonstrated in adult males and females of the same strain. Experiments indicated that testosterone does not act directly in the serum to expose Slp antigenic sites. Testosterone treatment of both males and females of strain C57BL/10JSf, which does not carry the gene for the presence of the Slp antigen, failed to stimulate the appearance of the antigen. Thus, the presence of Slp antigen in the serum is dependent on both the proper genotype and the presence of male hormone.Supported by U.S.P.H.S. Research Grant GM-15419, U.S.P.H.S. Training Grant 2T01-GM-00071 (H.C.P.), and U.S.P.H.S. Career Development Award K3-HE-24,980 (D.C.S.).     

Pregnancy rates, prenatal and postnatal survival of offspring, and litter sizes after reciprocal embryo transfer in DBA/2JHd, C3H/HeNCrl and NMRI mice

Success of embryo transfer is often a limiting factor in transgenic procedures and rederivation efforts, and depends on the genetic background of the donor and recipient strains used. Here we show that embryo transfer to DBA/2J females is possible, and present data on pre- and postnatal success rates after reciprocal embryo transfer using the inbred DBA/2J and C3H/HeN, and outbred NMRI strains. The highest embryo yield was achieved in outbred NMRI females, but embryo yields were similar in DBA/2J and C3H/HeN mice following superovulation despite poor estrus cycle synchronization in DBA/2J females. In-strain transfer of DBA/2J blastocysts (transfer of embryos to recipients from the same strain) resulted in pregnancy rates (57.1%) similar to those obtained following in-strain transfer of C3H/HeN (60.0%) and NMRI mice (83.3%), although the prenatal survival rate of blastocysts was low. Moreover, from the pups born only half survived the postnatal period after transfer of DBA/2J and C3H/HeN blastocysts to DBA/2J recipients. These problems were not observed when transferring NMRI-blastocysts to C3H/HeN and DBA/2J mothers. The number of blastocysts transferred also had a positive effect on the success of embryo transfer. In conclusion, C3H/HeN and DBA/2J females can be used as recipients for embryo transfer procedures for certain donor strains like NMRI, as one major determinant seems to be the genetic background of the embryos transferred. We also recommend to increase the number of DBA/2J blastocysts transferred, and to foster the DBA/2J pups to other DBA/2J mothers postnatally for in-strain transfer of DBA/2J mice.     

Immunohistochemical demonstration of androgen-binding protein in the rat prostatic gland.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.     

Serum protein polymorphisms in a Liberian population

Serum protein variations were studied in a Liberian population living in Buchanan town. Of the alpha 1-antitrypsin genes only M1 and M3 were polymorphic. The frequencies of the haptoglobin and Gc genes were in accordance with earlier known estimates in African populations. There was, however, a relatively low frequency of Hp 0 which may be related to the low malarial parasite prevalence in this group. The transferrin C2 gene was found in a significantly lower frequency among Liberians compared to European and Asiatic populations. A new transferrin variant was observed by isoelectric focusing. This variant could not be identified with conventional starch or polyacrylamide electrophoresis.     

Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.     

Structural and functional dissection of the Abp1 ADFH actin-binding domain reveals versatile in vivo adapter functions

Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.