An abundant androgen-regulated mRNA in the mouse kidney.

We have identified an abundant 20,000 dalton protein (KAP) by in vitro translation of male mouse kidney mRNA. This protein is synthesized in reduced amounts from female kidney mRNA. A KAP cDNA fragment was purified and used for nucleic acid hybridization studies. Females and castrated males have 10 and 200 fold lower levels, respectively, of KAP mRNA relative to males. The administration of testosterone to females or castrated males results in the induction of KAP mRNA to normal male levels. Testicular feminized (Tfm) mice have 3 fold lower levels of KAP mRNA relative to normal males and are not induced by testosterone. KAP mRNA is not found in significant amounts in tissues other than the kidney, and the KAP gene renatures with kinetics similar to single-copy DNA. With the rapidly expanding knowledge of mouse genetics, KAP should prove useful in determining genetic factors which regulate the inducibility and tissue specificity of a hormonally regulated gene.     

Genomic organization and DNA sequence of the mouse kidney androgen-regulated protein (KAP) gene

The gene for kidney androgen-regulated protein (KAP) is expressed under androgenic control in the epithelial cells of the renal cortical proximal tubules. However, there is an androgen-independent component of the expression of this gene that occurs specifically in the outermedullary S3 segments of the proximal tubules. In these cells, the KAP gene is estrogen responsive and its expression is dependent on pituitary function. As a first step in correlating its interesting cell-specific and hormonal regulation with the structure of the gene, the genomic organization of the KAP gene was described and sequence of the gene and the proximal 1 kb of 5'-flanking DNA was determined. Sequence motifs were identified in the 5'-flanking DNA that may function in the regulation KAP gene expression by androgen, estrogen, and pituitary glycoprotein hormones. The gene is present in a single copy in the mouse genome and is 3,807 nucleotides in length. It contains 4 exons of 120, 177, 63, and 251 nucleotides and three intervening sequences of 1,450, 126, and 1,620 nucleotides. The gene exhibits a high degree of a genetic polymorphism as revealed by comparison of restriction digests of DNA from two highly inbred strains, BALB/c and C57BL/6.     

Close linkage between mouse genes determining the two forms of complement component C6 and component C7, and cis action of a C6 Regulatory Gene

Two forms of mouse complement component C6, with molecular weights (M _rs) of 90 and 100 kilodaltons (kd), are present in the sera from certain inbred strains such as the CBA strain; other strains, such as the BALB/c and DBA/2 strains, have only the 90 kd C6A form. The present work was undertaken to determine whether the two M _r forms were the products of genes coding at separate loci. We screened sera from mice from a number of inbred strains by isoelectric focusing and found one strain, AKR, exhibiting allotypic structural variations of C6 forms. To distinguish the various types, we designated the 90 kd types from CBA and AKR mice C6A1 and C6A2, respectively, and the corresponding 100 kd types C6B 1 and C6B2, respectively. Mice possessing only one M _r form were all typed as C6A1. Results of breeding experiments strongly suggested that the two M _r forms of C6 are coded for at two closely linked loci. Sera from a number of inbred strains were also screened for a complement C7 polymorphism by means of isoelectric focusing and functional overlay. C7 from all strains, excepting the AKR strain, produced identical C7 band patterns. AKR C7 produced a unique band pattern, and results of breeding experiments with AKR and BALB/c mice showed the C6 and C7 loci to be closely linked. In addition, we identified a regulatory gene for C6 production. The gene apparently requires androgen to facilitate C6 production in the majority of strains. In these strains C6 activity is virtually absent from female sera. However, we observed moderate levels of C6 activity in sera from IS/Cam females, indicating that, in this strain, male physiological androgen levels are not necessary for C6 production. IS/Cam possess one form of circulating C6 which appears identical with BALB/c C6A1, and therefore IS/Cam mice differ from AKR mice at both the C6 structural and regulatory loci. These two strains were thus suitable for use in breeding experiments to determine the manner of action of the regulatory gene. Results showed that it acted in a cis manner.Abbreviations used in this paper M _r molecular weight - kd kilodaltons - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - IEF isoelectric focusing - Slp sex-limited protein - MHC major histocompatibility complex     

KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport

We cloned a new member of the murine brain kinesin superfamily, KIF3B, and found that its amino acid sequence is highly homologous but not identical to KIF3A, which we previously cloned and named KIF3 (47% identical). KIF3B is localized in various organ tissues and developing neurons of mice and accumulates with anterogradely moving membranous organelles after ligation of nerve axons. Immunoprecipitation assay of the brain revealed that KIF3B forms a complex with KIF3A and three other high molecular weight (approximately 100 kD)-associated polypeptides, called the kinesin superfamily-associated protein 3 (KAP3). In vitro reconstruction using baculovirus expression systems showed that KIF3A and KIF3B directly bind with each other in the absence of KAP3. The recombinant KIF3A/B complex (approximately 50-nm rod with two globular heads and a single globular tail) demonstrated plus end-directed microtubule sliding activity in vitro. In addition, we showed that KIF3B itself has motor activity in vitro, by making a complex of wild-type KIF3B and a chimeric motor protein (KIF3B head and KIF3A rod tail). Subcellular fractionation of mouse brain homogenates showed a considerable amount of the native KIF3 complex to be associated with membrane fractions other than synaptic vesicles. Immunoprecipitation by anti-KIF3B antibody-conjugated beads and its electron microscopic study also revealed that KIF3 is associated with membranous organelles. Moreover, we found that the composition of KAP3 is different in the brain and testis. Our findings suggest that KIF3B forms a heterodimer with KIF3A and functions as a new microtubule-based anterograde translocator for membranous organelles, and that KAP3 may determine functional diversity of the KIF3 complex in various kinds of cells in vivo.     

Nucleotide sequence of kidney androgen-regulated protein mRNA and its cell-specific expression in Tfm/Y mice

Kidney androgen-regulated protein (KAP) mRNA is an abundant renal mRNA that was originally identified by comparisons of the products of in vitro translation of poly(A) RNA from animals before and after androgen stimulation. KAP mRNA is 607 nucleotides long, excluding its poly(A) segment, and encodes a protein of 13,265 mol wt. A hydrophobic N-terminal domain forms a putative signal peptide of 18 amino acids, the cleavage of which results in a 103-amino acid mature protein with a molecular size of 11,297. The protein is highly negatively charged and contains regions of clustered Pro, Glu/Asp, Ser, and Thr residues that are associated with proteins with short half-lives. KAP mRNA is unusual in that it is expressed in two distinct regions of the kidney under different hormonal treatments. It is expressed throughout the cortex in the epithelial lining of the proximal tubules in response to androgen stimulation. After castration, only tubules in the outer stripe of the medulla express KAP mRNA. The androgen receptor-deficient Tfm/Y mutant strain exhibits KAP mRNA induction only in this juxtamedullary region after testosterone treatment. Expression of KAP mRNA in these cells is responsible for the relatively high basal levels of KAP mRNA in female and castrated male animals, and induction in these cells occurs by an androgen receptor (AR)-independent mechanism.     

Identification, partial purification and biochemical characterization of gamma-glutamyltranspeptidase present as a membrane component in skimmed milk and milk fat-globule membranes, and in mammary-tumour virus from the milk of infected mice.

The enzyme gamma-glutamyltranspeptidase was reproducibly found to be associated with mouse milk particles; it is present in milk fat-globule membranes and mouse mammary-tumour virus of infected Swiss mice, also in particles from the milk of uninfected mice. The enzymatic activities observed range among the highest reported for mammalian tissues. The enzyme was partially purified from mouse mammary-tumour virus, and from milk fat-globule membranes. The molecule requires the presence of detergents to remain soluble, behaves as a high molecular weight component, properties characterizing integral membrane proteins. Kinetics, and the effect of competitors as well as of specific inhibitors show this enzyme to be identical to the well-known kidney gamma-glutamyltranspeptidase ((gamma-glutamyl)-peptide:amino-acid gamma-glutamyltransferase, EC 2.3.2.2). Other oncornaviruses budding from cultured cells originally expressing the enzyme in their plasma membrane also incorporate the enzyme in their structure.     

Molecular cloning and analysis of DNA complementary to three mouse Mr = 68,000 heat shock protein mRNAs

The construction and isolation of three recombinant DNAs complementary to different mouse L-cell Mr = 68,000 heat shock protein (hsp68) mRNAs is described. cDNA libraries derived from heat-shocked mouse L-cell poly(A)+ RNA by the vector-linked primer strategy of cDNA synthesis and cloning of Okayama and Berg (Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170) were screened first with a Drosophila hsp70 heterologous probe and subsequently with a cDNA probe isolated from the first screening. Positive clones were assigned to one of three sets based on their restriction map, and the largest member of each group was chosen for further analysis. All three cDNAs hybrid-select mRNA for the mouse major heat shock protein (hsp68) as assayed by in vitro translation and hybridize preferentially to two heat shock-induced hsp68 mRNAs on Northern blots. The coding regions of the cDNAs are almost identical and closely resemble other HSP70 genes but the 3' untranslated regions diverge considerably. Differences in the lengths of the untranslated regions are responsible for the two different sized induced hsp68 mRNAs in mouse L-cells. The physical maps of these cDNA clones and the limited number of mouse genomic DNA fragments detected on Southern blots suggest that there are at least three closely related heat shock-inducible members of the mouse HSP70 gene family. None of the cloned cDNAs are derived from the two related cognate genes known to be present in the mouse genome.     

Unique patterns of androgen regulation of the expression of two genes in murine kidney

The kidneys of androgen stimulated mice exhibit a hypertrophic response but no hyperplasia or concomitant DNA replication. Androgens increase the expression of several genes in mouse kidney. The response of the beta-glucuronidase gene to testosterone in this tissue is characterized by a 1-2 day lag and relatively slow induction kinetics. The gene coding for kidney androgen-regulated protein (KAP) exhibits quite a different response to the hormone when compared on the basis of initial response to a given dose, dose required to produce maximal response, and apparent sensitivity to low levels of androgen-receptor complexes in renal nuclei. The analysis of the accumulation of the mRNAs produced by these two genes suggests that gene-specific differential sensitivity to androgen receptor complexes governs the development of the cellular male phenotype in this tissue.     

Kidney androgen-regulated protein interacts with cyclophilin B and reduces cyclosporine A-mediated toxicity in proximal tubule cells

The gene for kidney androgen-regulated protein (KAP) is the most abundant and specific gene expressed in mouse kidney proximal tubule cells, where it is tightly regulated by steroid and thyroid hormones in different tubule segments. Despite the cell-specific expression, strict regulatory mechanisms, and relative abundance, nothing is known of the function of its encoded protein, which does not exhibit known structural or functional domains, or homologies with other sequences in the data bases. We raised monoclonal antibodies against KAP, which specifically recognize a protein with an apparent molecular mass of 20 kDa in crude kidney homogenates, the distribution and regulation of which parallel that of its mRNA. To gain insight into its function, we performed a yeast two hybrid screen and determined that KAP specifically interacts with cyclophilin B. Furthermore, cyclosporine A (CsA)-treated mice exhibited a significant decrease in KAP levels, and tetracycline-controlled overexpression of KAP in stably transfected proximal tubule cells significantly decreased the toxic effects of CsA. Taken together, these results indicate a functional relationship among KAP-, cyclophilin B-, and CsA-mediated nephrotoxicity and suggest an important role of KAP in renal physiology, providing new data on the molecular mechanisms implied in the toxic effects of CsA.     

Amino-acid and cDNA nucleotide sequences of human Clara cell 10 kDa protein

A human lung cDNA expression library was screened by using a rabbit antiserum specific for a human Clara cell 10 kDa protein. The cDNA from two positive clones was sequenced by the dideoxy chain termination method. The nucleotide and primary amino-acid sequence deduced therefrom are presented. The N-terminal amino-acid sequence of the Clara cell 10 kDa protein, purified from bronchoalveolar lavage, was also determined. The deduced and experimentally determined sequences were identical where data for both were available. From the amino-acid composition, deduced and experimentally determined amino-acid sequences, it was determined that the 10 kDa protein in bronchoalveolar lavage consists of two identical 70-amino-acid long polypeptide chains joined by two cystine residues. The size of mRNA for the protein was found to be about 0.6 kb and the monomeric nascent protein, obtained by in vitro translation of lung mRNA was about 7.3 kDa in size. The 10 kDa protein recovered from bronchoalveolar lavage has 61% sequence identity with rabbit uteroglobin, the two proteins have common predicted secondary structures with marked surface differences when comparing predicted and actual structure determined by X-ray diffraction. The differences imply similarity of structure but, not identity of function.     

Translation of the L-species dsRNA genome of the killer-associated virus-like particles of Saccharomyces cerevisiae.

Virus-like particles containing the L (P1)-species of double-stranded RNA (dsRNA) were isolated from Saccharomyces cerevisiae, and the translational activity of the virus-like particle-derived dsRNA was analyzed in the wheat germ cell-free system. Denaturation of the dsRNA immediately prior to in vitro translation resulted in the synthesis of one major and at least three minor polypeptides, whereas undenatured dsRNA, as expected, did not stimulate [35S]methionine incorporation into polypeptides, but actually slightly inhibited endogenous activity. The major in vitro translation product of the denatured L-dsRNA was shown to be identical with the major L-dsRNA containing virus-like particle capsid polypeptide on the basis of three criteria: co-electrophoresis on sodium dodecyl sulfate polyacrylamide gels, immunoprecipitation, and tryptic peptide analysis. We have therefore established that the L-dsRNA genome encodes the major virus-like particle capsid polypeptide. This result adds considerable support to the hypothesis that the L-dsRNA genome acts as a helper genome to the smaller (1.6 x 10(6) dalton) M-dsRNA genome in killer strains of yeast by providing the M-dsRNA containing virus-like particles with their major coat protein.     

The mouse antibody heavy chain repertoire is germline-focused and highly variable between inbred strains

The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies.     

Determination of the expression of fish antifreeze protein (AFP) in 7th generation transgenic mice tissues and serum

In this study, the presence of antifreeze protein (AFP) gene expression through successive generations in transgenic mice carrying the chimeric gene construct of the coding sequence for the AFP protein from ocean pout was investigated. AFP transgenic hemizygote mice were used for AFP gene expression. AFP genome expressions in transgenic mice were analyzed by Western blotting, and tissue location of AFP protein was shown by immunohistochemical and immunofluorescence techniques. Seventh transgenic mice from the established founders demonstrated the expression of AFP in organs such as the skin, oviduct, lung, kidney and liver tissues and serum except for the heart. Our results demonstrate successful expression of AFP gene products in several tissues and serum of transgenic mice, the association of in vivo expressed AFP protein, for the first time. These results indicate that the coding sequence for the AFP protein gene (ocean pout type III AFP gene) could be integrated and stably transcribed and expressed in the 7th generation of transgenic mice. In conclusion transgenic mouse lines would be a good model for the cryostudy of AFP and for the determination of AFP roles in several organs and tissues.     

Detection of spatially regulated gene expression by hybridization histochemistry.

Steady state measurements of kidney androgen-regulated protein (KAP) mRNA suggested that KAP gene expression was unusually sensitive to low hormone-receptor levels. Two of the criteria used to reach this conclusion involved relative insensitivity to treatment with a competitive antiandrogen and a partial androgen response of the gene in Tfm/Y androgen receptor (AR) deficient mice. These data may indicate the ability of the KAP gene to respond to an extremely low level of androgen-AR complex or that the effect of androgens is, at least in part, indirect. Hybridization in situ revealed that KAP mRNA expression was restricted to proximal tubule epithelial cells in the juxtamedullary region of castrated animals rather than throughout the cortex as in intact males. Examination of sections of kidneys from Tfm/Y mice before and after testosterone (T) treatment revealed that only the juxtamedullary tubules expressed KAP mRNA and that T increased the level of KAP mRNA in these cells. Treatment of Tfm/Y animals with other steroids showed that beta-estradiol treatment mimicked the effect of T while dihydrotestosterone (DHT) had no effect. The facts that DHT and T both stimulate cortical expression of KAP mRNA in normal animals but DHT has no effect on the juxtamedullary cells in the Tfm/Y variant may indicate that the action of T is due to an estrogenic metabolite. Castrated, hypophysectomized males exhibited no KAP gene expression, while in the presence of T, expression was observed throughout the cortex as in intact males. These results clearly indicate the involvement of pituitary hormones in KAP gene expression in the juxtamedullary tubules. These studies have shown that the regulation of KAP gene expression in the mouse kidney is much more complex than originally believed. Future studies will further investigate the roles of estrogen and specific pituitary hormones in KAP gene expression.     

Purification and characterization of two mannan-binding lectins from mouse serum

Mannan-binding lectin (MBL) is a serum protein that activates the complement system after binding to glycoconjugates found on the surface of microorganisms. By molecular cloning two forms of MBL have been identified in the mouse (mMBL-A and mMBL-C), but only mMBL-A has been purified and characterized at the protein level. MBL-C has been termed the liver form of MBL. The present report describes the purification and characterization of mMBL-A and mMBL-C from serum. The two forms of mMBL could be separated both by ion-exchange and carbohydrate-affinity chromatography. The initial identification by immunochemical technique was confirmed by N-terminal amino-acid sequencing. Both proteins give bands corresponding to polypeptide chains of 28 kDa on SDS-PAGE in the reduced state, but mMBL-A migrated more rapidly than mMBL-C in acid/urea-PAGE, in accordance with the calculated pIs. Both forms mediated activation of complement component C4 in mannan-coated microtiter wells. MBL-A showed a higher affinity for d -glucose and alpha-methyl-d -glucose then did MBL-C. Serum concentrations of mMBL-A in laboratory strains and wild mice were found to vary from 5 to 80 microg/ml, with wild mice tending to show higher levels than laboratory strains.     

The rat kidney acylase I, characterization and molecular cloning. Differences with other acylases I.

The soluble acylase I from rat kidney was purified to homogeneity using a five-step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-L-methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224-bp open reading frame encoding a protein consisting of 408 amino-acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.     

Wide Distribution of Mitochondrial Genome Rearrangements in Wild Strains of the Cultivated Basidiomycete Agrocybe aegerita

We used restriction fragment length polymorphisms to examine mitochondrial genome rearrangements in 36 wild strains of the cultivated basidiomycete Agrocybe aegerita, collected from widely distributed locations in Europe. We identified two polymorphic regions within the mitochondrial DNA which varied independently: one carrying the Cox II coding sequence and the other carrying the Cox I, ATP6, and ATP8 coding sequences. Two types of mutations were responsible for the restriction fragment length polymorphisms that we observed and, accordingly, were involved in the A. aegerita mitochondrial genome evolution: (i) point mutations, which resulted in strain-specific mitochondrial markers, and (ii) length mutations due to genome rearrangements, such as deletions, insertions, or duplications. Within each polymorphic region, the length differences defined only two mitochondrial types, suggesting that these length mutations were not randomly generated but resulted from a precise rearrangement mechanism. For each of the two polymorphic regions, the two molecular types were distributed among the 36 strains without obvious correlation with their geographic origin. On the basis of these two polymorphisms, it is possible to define four mitochondrial haplotypes. The four mitochondrial haplotypes could be the result of intermolecular recombination between allelic forms present in the population long enough to reach linkage equilibrium. All of the 36 dikaryotic strains contained only a single mitochondrial type, confirming the previously described mitochondrial sorting out after cytoplasmic mixing in basidiomycetes.