Assignment of the gene encoding the catalytic subunit Cβ of CAMP-dependent protein kinase to the p36 band on chromosome

Summary A cDNA for the human catalytic subunit (C) of cAMP-dependent protein kinase (PKA) has been cloned from a testis cDNA library. In the present study, we have determined the chromosomal localization of this gene using a cDNA for C as a probe. Southern blot analysis of genomic DNA from human/mouse cell hybrids revealed that the presence or absence of a 20-kbXbaI fragment, which hybridized with the C probe, was concordant with the presence of human chromosome 1.In situ hybridization to metaphase chromosome confirmed the somatic cell hybrid data and regionally mapped the C gene of PKA to the p36 band on chromosome 1.     

Molecular cloning of a tissue-specific protein kinase (C gamma) from human testis--representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase

Two different mammalian genes for the catalytic subunit (C) of cAMP-dependent protein kinase have previously been characterized (C alpha, C beta). In the present study, we report the molecular cloning of a third isoform of C, from a human testis cDNA library, as well as the isolation of human cDNAs for C alpha and C beta. This third form of C, which we will designate C gamma, is clearly derived from a distinct gene and shows a tissue-specific expression. A close evolutionary relation between C gamma and C alpha was suggested by nucleotide homologies (86% inside the open reading frame, 81% in the 3'-untranslated region). Thus, the C gamma cDNA cross-hybridized with the 2.8 kilobase (kb) C alpha mRNA, present at high levels in most human tissues, as well as with a 1.8 kb C gamma-specific mRNA, which was only found at detectable levels in human testis. However, at the amino acid level, C alpha and C beta showed a close relationship (93% homology), whereas C gamma diverged significantly from both C alpha (83%) and C beta (79%). Taken together with the tissue-specific expression of C gamma, this suggests a pressure on C gamma during evolution, acting to modulate it in a functionally specific way. Certain amino acid substitutions make C gamma a distinct member of the cAMP-dependent subfamily of protein kinases, and suggest that C gamma may be distinct in its protein substrate specificity or its interaction with the different regulatory subunits.     

Molecular cloning and chromosomal mapping of the human gene for the testis-specific catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A).

A cDNA for an alternatively spliced variant of the testis-specific catalytic subunit of calmodulin dependent protein phosphatase (CaM-PrP) was cloned from a human testis library. The nucleotide sequence of 2134 base pairs (bp) encodes a protein of 502 amino acids (Mr approximately 57,132) and pI 7.0. The cDNA sequence differs from the murine form of this gene by a 30 bp deletion in the coding region, the position of which matches those in the two other genes for the catalytic subunit. These data indicate that this alternative splicing event arose prior to the divergence of the three genes. The deduced sequence of the human protein is only 88% identical to the homologous murine form, in striking contrast to the other two CaM-PrP catalytic subunits which are highly conserved between mouse and human (approximately 99%); this indicates a more rapid rate of evolution for the testis-specific gene. Analysis of Southern blots containing DNA from human-hamster somatic cell hybrids show that the gene is on human chromosome 8.     

Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32----qter

The nicotinic acetylcholine receptor of skeletal muscle (CHRN in man, Acr in mouse) is a transmembrane protein composed of four different subunits (alpha, beta, gamma, and delta) assembled into the pentamer alpha 2 beta gamma delta. These subunits are encoded by separate genes which derive from a common ancestral gene by duplication. We have used a murine full-length 1,900-bp-long cDNA encoding the gamma subunit subcloned into M 13 (clone gamma 18) to prepare single-stranded probes for hybridization to EcoRI-digested DNA from a panel of human x rodent somatic cell hybrids. Using conditions of low stringency to favor cross-species hybridization, and prehybridization with rodent DNA to prevent rodent background, we detected a single major human band of 30-40 kb. The pattern of segregation of this 30-40 kb band correlated with the segregation of human chromosome 2 within the panel and the presence of a chromosomal translocation in the distal part of the long arm of this t(X;2)(p22;q32.1) chromosome allowing the localization of the gamma subunit gene (CHRNG) to 2q32----qter. The human genes encoding the gamma and delta subunits have been shown to be contained in an EcoRI restriction fragment of approximately 20 kb (Shibahara et al., 1985). Consequently, this study also maps the delta subunit gene (CHRND) to human chromosome 2q32.1----qter. In the mouse, the Acrd and Acrg genes have been shown to be linked to Idh-1, Mylf (IDH1 and MYL1 in humans, respectively) and to the gene encoding villin on chromosome 1. Interestingly, we have recently localized the human MYL1 gene to the same chromosomal fragment of human chromosome 2. These results clearly demonstrate a region of chromosomal homoeology between mouse chromosome 1 and human chromosome 2.     

A novel isoform of human cyclic 3',5'-adenosine monophosphate-dependent protein kinase, c alpha-s, localizes to sperm midpiece

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence comparison revealed similarity to an ovine C alpha variant characterized by protein purification and micropeptide sequencing, C alpha-s, identifying the cloned human cDNA as the C alpha-s isoform. The C alpha-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The C alpha-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C alpha-s-specific antibody indicated that C alpha-s was localized in the midpiece region of the spermatozoon. The majority of C alpha-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the C alpha-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RI alpha, RII alpha, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target C alpha-s. This novel C alpha-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.     

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.     

Chromosomal assignment of genes encoding the alpha, beta, and gamma subunits of human complement protein C8: identification of a close physical linkage between the alpha and the beta loci

The eighth component of human complement (C8) is a serum protein containing three nonidentical subunits (alpha, beta, gamma) that are arranged as a disulfide-linked alpha-gamma dimer and a noncovalently associated beta chain. In earlier genetic studies, electrophoretic analysis of C8 protein polymorphisms revealed several allelic variants of alpha-gamma and beta. These were governed by separate loci designated C8A and C8B for alpha-gamma and beta, respectively. Genetic linkage analyses indicated that these loci were linked to each other and to chromosome 1 marker loci PGM1 and Rh, but it was unclear at the time if C8A was a single locus coding for a single-chain precursor form of alpha-gamma or if separate loci existed for alpha and gamma. Since evidence now indicates that alpha, beta, and gamma are encoded by separate genes, cDNA probes corresponding to each subunit were used to make direct assignments of the individual loci. Analysis of somatic cell hybrids revealed that only the alpha and beta loci are located on chromosome 1. Parallel analysis of genomic DNA digests using 5' and 3'-specific cDNA probes showed they are physically linked (less than 2.5 kb) and oriented 5' alpha-beta 3'. Further probing of the hybrid panel revealed that gamma is located on chromosome 9q. Thus, the observed genetic linkage of alpha-gamma to beta must be determined solely by alpha. In accordance with these findings, the C8 loci should now be designated C8A, C8B, and C8G for alpha, beta and gamma, respectively.     

Decreased expression of the type I isozyme of cAMP-dependent protein kinase in tumor cell lines of lung epithelial origin

A spontaneous transformant derived from a mouse lung epithelial cell line exhibited decreased cAMP-dependent protein kinase (PKA) activity. DEAE column chromatography demonstrated that this was caused by specific loss of the type I PKA isozyme (PKA I). Western immunoblot analysis indicated that indeed several mouse lung tumor-derived cell lines and spontaneous transformants of immortalized, nontumorigenic lung cell lines contained less PKA I regulatory subunit (RI) protein than normal cell lines. PKA II regulatory subunit protein differed only slightly among cell lines and showed no conspicuous trend between normal and neoplastic cells. The decrease in RI was apparently concomitant with decreased catalytic (C) subunit levels in neoplastic cells since no free catalytic subunit activity was detected by DEAE chromatography. Northern blot analysis using RI alpha and C alpha cDNA probes showed that the levels of RI alpha and C alpha mRNAs paralleled their intracellular protein concentrations; neoplastic cell lines contained significantly less RI alpha and C alpha mRNAs than the normal cell line. The decreased expression of both RI and C subunits therefore results in a net decrease of PKA I in neoplastic lung cells, an isozymic difference which may account for the differential effects of cAMP analogs on cell growth and differentiation in normal and neoplastic cells.     

Cloning, characterization, and mapping of the gene encoding the human G protein gamma 2 subunit

G proteins play vital roles in cellular responses to external signals. The specificity of G protein-receptor interaction is mediated mostly by the gamma-subunit and the individual members of the gamma-subunit multigene family would hence be expected to each have a particular expression profile. In an experiment designed to isolate genes expressed predominantly in human testis we identified a cDNA fragment corresponding to the gamma2 gene. Although the protein sequence of the gamma2 subunit has previously been published, the cDNA sequence, expression pattern, genomic structure, and localisation of the human GNG2 gene have not been described. We report the complete sequence of the GNG2 cDNA which is 1066 bp long and contains an open reading frame encoding a protein of 71 amino acids. This protein is 100% homologous to the bovine, mouse, and rat G protein gamma2 subunit. The gene structure is very similar to that of other Ggamma-subunit genes in that there are two introns, one located in the 5' UTR and the other within the ORF. We show that this gene is expressed in a range of foetal tissues as well as adult testis, adrenal gland, brain, white blood cells and lung but not in adult liver, muscle, sperm, prostate gland nor in the testes of two different infertile patients. There is evidence that GNG2 is expressed in malignant tissues. Using two independent methods, we have mapped the human GNG2 gene to chromosome 14q21.     

Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1).

The gene (HSD-1) coding a human sperm membrane protein (hSMP-1) was isolated from a human testis cDNA expression library using antibodies found in the serum of an infertile woman. HSD-1 was localized to a single locus on chromosome 9 and assigned to band 9p12-p13 by fluorescent in situ hybridization (FISH) mapping and DAPI (4,6-diamidino-2-phenylindole) banding, using rat/human somatic cell hybrids and metaphase chromosomes of human lymphocytes. In rescreening a testis lambdagt10 cDNA expression library, the full-length cDNA (HSD-1) and several truncated cDNAs with heterologous regions were isolated from positive clones. The heterology consisted of deletion, insertion and alteration of the 5'-end. These heterologous truncated fragments may be produced by alternative splicing of mRNAs. Two recombinant prokaryotic expression vectors were constructed with one of the heterologous fragment (clone #26) with and without the alternative 5'-end. Escherichia coli transfected with the construct containing the alternative 5'-end failed to produce the recombinant product, whereas those transfected with the vector lacking the 5'-end produced hSMP-1. DNASIS analysis of the structure of #26 mRNA suggests that the 5'-end has a stable secondary configuration that may maintain the mRNA in an inactivated state, whereby hindering its translation and preventing the expression of the gene.     

The NDUFS4 nuclear gene of complex I of mitochondria and the cAMP cascade

Results of studies on the role of the 18 kDa (IP) polypeptide subunit of complex I, encoded by the nuclear NDUFS4 gene, in isolated bovine heart mitochondria and human and murine cell cultures are presented.The mammalian 18 kDa subunit has in the carboxy-terminal sequence a conserved consensus site (RVS), which in isolated mitochondria is phosphorylated by cAMP-dependent protein kinase (PKA). The catalytic and regulatory subunits of PKA have been directly immunodetected in the inner membrane/matrix fraction of mammalian mitochondria. In the mitochondrial inner membrane a PP2Cgamma-type phosphatase has also been immunodetected, which dephosphorylates the 18 kDa subunit, phosphorylated by PKA. This phosphatase is Mg(2+)-dependent and inhibited by Ca(2+). In human and murine fibroblast and myoblast cultures "in vivo", elevation of intracellular cAMP level promotes phosphorylation of the 18 kDa subunit and stimulates the activity of complex I and NAD-linked mitochondrial respiration.Four families have been found with different mutations in the cDNA of the NDUFS4 gene. These mutations, transmitted by autosomal recessive inheritance, were associated in homozygous children with fatal neurological syndrome. All these mutations destroyed the phosphorylation consensus site in the C terminus of the 18 kDa subunit, abolished cAMP activation of complex I and impaired its normal assembly.