Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Stop codon”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,333 records · Page 74Linked to original sources
PubMed1993

[The point mutation of pre-C region HBV-DNA in sera from chronic hepatitis B].

To clarify the relationship between a point mutation of HBV-DNA Pre-C region and the serological feature of chronic hepatitis B, we determined the Pre-C region sequence of HBV-DNA obtained sera of 39 patients with chronic hepatitis B. The screening of the gene arrangement of Pre-C region of HBV-DNA was performed by a direct sequence method amplifying HBV-DNA by polymerase chain reaction methods. In 22 of HBeAg positive cases, a mutant type (the 28th codon changed from TGA to TAG: stop codon) was found in only one case. The other hand, In 17 of HBeAg negative cases, it was found in 6 cases with fluctuating ALT and DNA-polymerase levels. We concluded that mutant viral infections could be the main cause of HBeAg negative cases with fluctuating ALT and DNA-polymerase levels.

Adult↗
PubMed1996

[Trace elements as activation factors of enzymes: characteristic enzymes and their activation mechanisms].

We here describe activation mechanisms of three characteristic metalloenzymes by metals. The copper-containing amine oxidase contains a topaquinone residue at the active site. The inactive precursor form of the enzyme, which contains a tyrosine residue at the position corresponding to topaquinone of the matured protein, was isolated and converted to the matured form by incubation with cupric ions through self-processing of the protein. The selenocysteine residue of selenium-containing formate dehydrogenase is synthesized cotranslationally through a process directed by a UGA codon that normally functions as a stop codon. We describe the mechanism of recognition of UGA and specific incorporation of selenium into the protein. Vanadium-containing haloperoxidases afford stable apo forms lacking vanadium. Reconstitution of holo forms by vanadium ions are also described.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia.

Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II). We investigated the b5R gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations. The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele. From this mutant allele, the product without the catalytic portion of the enzyme is generated. The second one was a missense mutation at codon 95 (CCC-->CAC) in the other allele with the result that Pro changed to His within the flavin adenine dinucleotide (FAD)-binding domain of the enzyme. To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coll. The mutant enzyme showed less catalytic activity, less thermostability, and a greater susceptibility to trypsin than did the normal counterpart. The absorption spectrum of the mutant enzyme in the visual region differed from that of the wild-type. These results suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme. The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient.

Alleles↗
PubMed1996

Bernard-Soulier syndrome with severe bleeding: absent platelet glycoprotein Ib alpha due to a homozygous one-base deletion.

Bernard-Soulier syndrome is a rare congenital platelet disorder that affects a surface membrane adhesion receptor, glycoprotein (GP) Ib-V-IX. Both the genetic defects and the bleeding diatheses associated with the syndrome are heterogeneous due, in part, to the complexity of the involved receptor which consists of four different members, GPs: Ib alpha-Mr 143 K (contains the von Willebrand factor-binding site), Ib beta-Mr 22 K, V-Mr 83 K and IX-Mr 20 K. We studied a kindred that includes a 40 year-old man with severe Bernard-Soulier syndrome: life-threatening gastrointestinal bleeding, thrombocytopenia, giant platelets and absent ristocetin-dependent platelet aggregation. By Southern blotting, PCR amplification/sequencing, hetero-duplex analysis, and allele-specific oligonucleotide hybridization, the Ib-V-IX genes were analyzed, and the molecular genetic defect was defined as a one-base deletion in the GPIb alpha gene, involving an adenine of codon 19. The mutation, K19R, homozygous in the propositus and heterozygous in the available unaffected relatives, leads to a frame shift in codons 19-21 and a premature stop codon after codon 21. No functional GPIb alpha can be produced from the mutant allele, implying that the platelets of the affected patient lack all GPIb alpha. Within the spectrum of Bernard-Soulier syndrome, this patient's disorder exemplifies a severe or "classic" extreme; an "experiment of Nature" that illustrates the effect of a complete deficiency of the ligand-binding chain (GPIb alpha) of the GPIb-V-IX receptor.

Adult↗
PubMed1997

Solution structure of SECIS, the mRNA element required for eukaryotic selenocysteine insertion--interaction studies with the SECIS-binding protein SBP.

Selenocysteine, a selenium-containing analog of cysteine, is found in the prokaryotic and eukaryotic kingdoms in active sites of enzymes involved in oxidation-reduction reactions. This aminoacid is cotranslationally incorporated at UGA codons which usually act as translation stop codons. In eukaryotes, decoding of selenocysteine necessitates the participation of the selenocysteine insertion sequence (SECIS), an element lying in the 3'-untranslated region of selenoprotein mRNAs. A detailed experimental study of the secondary structures of the SECIS elements of rat and human type 1 iodothyronine deiodinases and rat glutathione peroxidase was performed. Enzymatic and chemical structure probing led us to propose a secondary structure model, supported by sequence comparison of 23 SECIS mRNAs. The secondary structure model revealed the existence of a novel type of RNA motif composed of four consecutive non-Watson-Crick base-pairs. Using gel shift experiments, we identified in several mammalian cell type extracts the protein SBP, for SECIS-binding protein, that specifically recognizes the iodothyronine deiodinases and glutathione peroxidase SECIS elements. The structural model that we derived for the SECIS RNAs discloses RNA features possibly implicated in the binding of SBP and/or SECIS function.

Animals↗
PubMed1997

Disease expression in X-linked retinitis pigmentosa caused by a putative null mutation in the RPGR gene.

PURPOSE: To determine the disease expression in X-linked retinitis pigmentosa (XLRP) caused by a putative null mutation in the RPGR (retinitis pigmentosa GTPase regulator) gene. METHODS: In a family with XLRP, haplotype analysis was performed with polymorphic microsatellite markers from the Xp chromosomal region, and genomic polymerase chain reaction sequencing was used to identify sequence variations in the RPGR gene. Hemizygotes and heterozygotes were evaluated clinically and with visual function tests. Optical coherence tomography (OCT) was performed on heterozygotes. Postmortem donor retinas from a heterozygote were examined by microscopy and immunocytochemistry. RESULTS: X-linked inheritance was confirmed by haplotype analysis using Xp markers. Sequence analysis of the RPGR gene identified a single base pair change, a G-->T transversion, that converts codon 52 GGA (Gly) to TGA (stop codon); the mutation segregates with the disease. A hemizygote in the third decade of life had barely measurable rod function and severely impaired cone function that diminished further over a 7-year interval. Heterozygotes varied in degree of disease expression from mild to severe. Perimetry showed loci with normal rod and cone sensitivity interspersed with loci having either equal rod and cone dysfunction or rod > cone dysfunction. Electroretinographic photoreceptor responses had equal reductions in rod and cone maximal amplitude. OCT cross sectional reflectance images of retinal regions with severe dysfunction showed reduced thickness of the retina and retinal pigment epithelium-choriocapillaris (RPE-CC) complex and increased reflections posteriorly. Regions with mild dysfunction showed similar OCT findings but with preserved retinal thickness. Retinal histopathology in a heterozygote revealed loss of photoreceptors throughout, with retention of only a few islands of cones with tiny or absent outer segments and rods lacking outer segments. CONCLUSIONS: This RPGR gene mutation, in its mildest expression in heterozygotes, causes a relatively equal disturbance of rod and cone photoreceptor function. Detectable structural change by OCT at the level of the RPE-CC can be present in patches of retina with minimal functional disturbance. More advanced disease stages in heterozygotes show greater rod than cone dysfunction, and the end stage in hemizygotes and heterozygotes is that of typical RP, with only barely detectable cone function from residual cones in a thinned retina with abnormal RPE and choriocapillaris.

Adult↗
PubMed1997

Production of rabbit polyclonal antibody against apobec-1 by genetic immunization.

Circulating apolipoprotein B (apoB) exists in two forms; apoB-100 and apoB-48. ApoB-48 is a truncated form of apoB resulting from RNA editing. The editing enzyme, called apobec-1, converts a cytidine (C) at nucleotide 6666 in apoB 100 mRNA to a uridine (U) and changes a CAA codon to an in-frame stop codon, UAA. We have produced a specific rabbit polyclonal antiserum against apobec-1 by genetic immunization. The cDNA of mouse apobec-1 was inserted downstream and in-frame at the BamH I site in the last exon of human growth hormone cDNA driven by a cytomegalovirus promoter. This plasmid was injected together with another plasmid expressing granulocyte macrophage colony-stimulating factor into the thigh muscles of a rabbit. The resulting antiserum demonstrated high specificity on Western blots, and inhibited the apoB mRNA editing activity of mouse liver extract in a dose-dependent manner. This report demonstrates that DNA immunization is a powerful technique that can be readily applied to other sparse or difficult-to-purify proteins in lipid metabolism.

APOBEC-1 Deaminase↗
PubMed1998

Cloning and sequencing of the celA gene encoding CMCase of Erwinia carotovora subsp. carotovora LY34.

The phytopathogenic Erwinia carotovora subsp. carotovora LY34 secretes multiple isozymes of the plant cell wall-disintegrating enzyme, endoglucanases. Genomic DNA from Ecc LY34 was digested with Sau3AI and ligated into the BamHI site of pBluescript II SK+. One of the E. coli clones containing a Sau3AI fragment of Ecc genomic DNA hydrolyzed carboxymethyl cellulose and was shown to contain the 2.2 kb BamHI restriction fragment, which was subcloned to generate pLYCA100 named as celA. The structural organization of a celA gene encoding 387 amino acids consists of an open reading frame (ORF) of 1161 bp starting with an ATG start codon and followed by a TAA stop codon. CelA protein contained a typical catalytic domain, interdomain, cellulose binding domain, and prokaryotic signal peptide of 32 amino acids. Since the deduced amino acid sequences of CelA protein was very similar to those of CelV of Erwinia carotovora subsp. carotovora SCC3193 enzyme and to those of CelN of Erwinia atroceptica enzyme, it belongs to the cellulase family 5. The apparent molecular mass of CelA protein was calculated to be 39 kDa by carboxymethylcellulose-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). Activity staining of carboxymethyl cellulase (CMCase) in sodium dodecyl sulfate polyacrylamide gel containing 0.1% CMC revealed that the cloned isozyme comigrated with a corresponding isozyme produced by Ecc LY34. The CelA had a calculated pI of 5.42. The optimum pH was 7 and the optimum temperature was about 45 degrees C.

Amino Acid Sequence↗
PubMed1998

A novel mutation in the D3 domain of von Willebrand factor markedly decreases its ability to bind factor VIII and affects its multimerization.

In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient's vWF gene showed, at heterozygous state, a G-->A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C-->T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient's vWF. The monoclonal antibody 31H3 against D' domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D' domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.

Amino Acid Substitution↗
PubMed2001

Analysis of the roles of tRNA structure, ribosomal protein L9, and the bacteriophage T4 gene 60 bypassing signals during ribosome slippage on mRNA.

A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames. In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame. Mutations affecting ribosomal protein L9 or tRNA(Gly)(2), the tRNA that decodes GGA, alter the efficiency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing signals and mutant translational components on -1 frameshifting at G GGA and hopping over a stop codon immediately flanked by two GGA glycine codons (stop-hopping). Mutant variants of tRNA(Gly)(2) that impair bypassing mediate stop-hopping with unexpected landing specificities, suggesting that these variants are defective in ribosomal P-site codon-anticodon pairing. In a direct competition between -1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of -1 frameshifting without substantially impairing the ability of mutant tRNA(Gly)(2) variants to re-pair with the mRNA by sub-optimal pairing. These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing. Two of the bypassing signals, a cis-acting nascent peptide encoded by the first open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing.

Anticodon↗
PubMed1988

Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2.

A 6.5-kilobase HindIII fragment that specifies the proteolytic activity of Streptococcus cremoris Wg2 was sequenced entirely. The nucleotide sequence revealed two open reading frames (ORFs), a small ORF1 with 295 codons and a large ORF2 containing 1,772 codons. For both ORFs, there was no stop codon on the HindIII fragment. A partially overlapping PstI fragment was used to locate the translation stop of the large ORF2. The entire ORF2 contained 1,902 coding triplets, followed by an apparently rho-independent terminator sequence. The inferred amino acid sequence would result in a protein of 200 kilodaltons. Both ORFs have their putative transcription and translation signals in a 345-base-pair ClaI fragment. ORF2 is preceded by a promoter region containing a 15-base-pair complementary direct repeat. Both the truncated 33- and the 200-kilodalton proteins have a signal peptide-like N-terminal amino acid sequence. The protein specified by ORF2 contained regions of extensive homology with serine proteases of the subtilisin family. Specifically, amino acid sequences involved in the formation of the active site (viz., Asp-32, His-64, and Ser-221 of the subtilisins) are well conserved in the S. cremoris Wg2 proteinase. The homologous sequences are separated by nonhomologous regions which contain several inserts, most notably a sequence of approximately 200 amino acids between the His and Ser residues of the active site.

Amino Acid Sequence↗
PubMed1997

Mutations of the Ki-ras, p53 and APC genes in adenocarcinomas of the human small intestine.

In contrast to the origins of colorectal carcinomas, the mechanisms of carcinogenesis in the small intestine remain unclear. We therefore analyzed the mutational status of the Ki-ras, p53, and adenomatous polyposis coli (APC) genes in primary carcinomas of the small intestine and compared the mutation patterns with those established for colorectal cancers. DNA was extracted from 15 formalin-fixed, paraffin-embedded lesions. Codons 12, 13 and 61 of the Ki-ras gene, exons 5-8 of the p53 gene, and codons 1268-1569, which contain the mutation cluster region (MCR) of the APC gene, were amplified by means of PCR, subcloned and sequenced. Mutations of the Ki-ras and p53 genes were observed in 8 (53.3%) and 4 lesions (26.7%), respectively. The mutational frequency of the Ki-ras gene in the present series of small intestinal carcinomas was similar, while that of the p53 gene was slightly lower than the reported frequencies for colorectal carcinomas. Only one case showed a mutation of the APC gene, involving an insertional mutation of an adenine at codons 1554-1556 with formation of a stop codon immediately downstream. Since the occurrence of an APC mutation is considered an early event in colorectal carcinogenesis, our findings indicating an extremely low frequency of such changes in and around the MCR suggest that carcinomas of the small intestine arise via a genetic pathway distinct from that involved in the development of carcinomas of the colorectum.

Adult↗
PubMed1998

A novel frameshift mutation 840delA and a novel polymorphism D203A in the steroidogenic acute regulatory protein gene in a Japanese patient with congenital lipoid adrenal hyperplasia. Mutations in brief no. 117. Online.

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomalrecessive disorder characterized by impaired production of all steroids including glucocorticoids, mineralocorticoids and sexsteriods. It has recently been reported that mutations in the steriodogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the StAR gene in a Japanese patient with CLAH. The patient was revealed to be a compound heterozygote bearing a nonsense mutation Q258X, changing codon 258 (CAG) encoding Gln to the stop codon TAG, and a novel framshift mutation 840delA resulting from deletion of one of the three adenosines normally present in codon 238 (AAA), thus leading to a frameshift after codon 237 (Thr) in the StAR gene. The patient was also revealed to be homozygous for a novel missense point mutation D203A, changing codon 203 (GAC) encoding Asp to GCC encoding Ala in the StAR gene. To elucidate the significance of the D203A mutation, we analyzed the StAR gene sequence in twenty normal subjects, and found that all of them were homozygous for the D203A mutation, indicating that the D203A mutation is an innocent polymorphism. In conclusion, we have identified a novel frameshift mutation 840delA which seems to cause 840delA and the first polymorphism D203A in the human StAR gene.

Adrenal Hyperplasia, Congenital↗
PubMed1998

Cloning and sequencing of cDNA encoding for a human sperm antigen involved in fertilization.

A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-lambda gt11 expression library by using antibodies to human sperm surface antigens belonging to 14-18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5' and 139-bp 3' noncoding regions. The translated protein has a calculated molecular weight of approximately 19 kD, and has two casein kinase II (CK-2) sites at aa 94-97 and 149-152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935-76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of approximately 20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The approximately 20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans.

Amino Acid Sequence↗
PubMed2005

Possible association between autism and variants in the brain-expressed tryptophan hydroxylase gene (TPH2).

We report a possible association between autism in our sample and a recently described brain-expressed tryptophan hydroxylase gene (TPH2). The well-replicated involvement of the serotonin neurotransmitter system in autism has stimulated interest in many genes in the serotonin pathway as possible candidates for mutations leading to autism susceptibility. Serotonin synthesis is controlled by the rate-limiting enzyme tryptophan hydroxylase. A mouse study of the original tryptophan hydroxylase gene (TPH1) and the new isoform (TPH2) showed that while TPH1 is primarily expressed peripherally, TPH2 is found exclusively in brain tissue. We searched for human sequence variants in 6,467 nucleotides covering all 11 exons of TPH2, and also 248 nucleotides upstream of the start codon, and 935 nucleotides downstream of the stop codon. Eighteen variants were characterized in 88 subjects with autism studied at our two centers, and 95 unrelated control subjects. Using a model-free association method and empirical P value estimation, two variants showed frequency differences between autism and control subjects (P = 0.01 for a T-G variant in intron 1, and P = 0.02 for a A-T variant in intron 4). A haplotype including these variants showed slightly increased significance (P = 0.005). Further investigation of clinical phenotypes showed a possible association between presence of the variants at these two SNPs and higher scores on the Autism Diagnostic Interview (ADI) domain describing repetitive and stereotyped behaviors (P = 0.007). We conclude that TPH2 may play a modest role in autism susceptibility, perhaps relating specifically to repetitive behaviors, pending replication of this result.

Autistic Disorder↗
PubMed1990

Defects in the precore region of the HBV genome in patients with chronic hepatitis B after sustained seroconversion from HBeAg to anti-HBe induced spontaneously or with interferon therapy.

Hepatitis B virus DNA clones were propagated from sera of six patients with chronic hepatitis B who seroconverted from HBeAg to antibody to HBeAg either spontaneously or after administration of alpha-interferon. Defects in the precore region blocking synthesis and secretion of HBeAg were detected in all 46 hepatitis B virus DNA clones from three patients who remained positive for antibody to HBeAg and in whom hepatitis resolved. Defective clones had point mutations from guanine to adenine at nucleotide 83 in the precore region, converting codon 28 from tryptophan (TGG) to a stop codon (TAG). In contrast, this defect was not found in any of 39 hepatitis B virus DNA clones from three patients who seroconverted to antibody to HBeAg but then redeveloped HBeAg with reactivation of hepatitis. Using these results, the G-to-A point mutation at nucleotide 83 in the precore region would predict sustained positivity for antibody to HBeAg and remission of hepatitis in patients who have seroconverted either spontaneously or with interferon therapy.

Adult↗
PubMed1992

p53 gene mutations in human endometrial carcinoma.

Although carcinoma of the uterine endometrium is the most frequently diagnosed malignancy of the female reproductive tract, the molecular genetic features of this tumor have yet to be described in significant detail. Since mutations of the p53 tumor suppressor gene are the single most common genetic alteration found in human malignancies, we examined the hypothesis that p53 mutations occur in human endometrial carcinoma. Sequencing analysis of exons 5-8 revealed point mutations in 3 of 21 (14%) tumors; one mutation was an unusual single-base insertion at codons 176-177, resulting in a premature stop codon, whereas the other two were CGG----TGG transitions at codon 248. Two of these tumors showed reduction to homozygosity at the p53 allele, but one tumor apparently retained heterozygosity. These data indicate that p53 mutations occur in human endometrial carcinoma, although relatively infrequently, and that loss of the normal p53 allele does not necessarily occur with point mutation of the p53 gene in this tumor type.

Alleles↗
PubMed2001

Cloning and sequencing of cDNA encoding for a novel human testis-specific contraceptive vaccinogen: role in immunocontraception.

Sperm-specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the subtractive cDNA hybridization technology, the present study was conducted to obtain a human sperm-specific antigen. The 32P-labeled single stranded cDNA of human testis, subtracted with poly(A)+ RNA of human peripheral white blood cells, was used to screen the human testis cDNA-ZAP II library. The putative positive clones were further screened for binding with the solubilized human oocyte zona pellucida preparation (HZP). After screening 10(7) colonies, one positive clone, designated contraceptive vaccinogen (CV), was obtained. It had an insert of approximately 1.3 kb, that was cloned and sequenced. The sense strand was identified by using the in vitro transcription and translation procedures, and the full-length sequence was obtained by using the 5' rapid amplification of 5' -cDNA ends (5'-RACE) procedure. The full-length CV cDNA has an ORF of 312 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 35 and the stop codon TAA, at nt 959. The translated protein has a calculated molecular mass of 35.3 kD and four potential N-linked glycosylation and several phosphorylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a membrane-anchored peptide. Extensive computer search in the database did not find any homology of existing sequences with CV both for nt and aa. Northern blot analysis indicated the human testis-specific expression of CV antigen. The coding region of CV cDNA was subcloned into pET22b(+) vector and expressed. The expressed recombinant (r)CV protein had a molecular size of approximately 44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV antibodies recognized the rCV, and a cognate antigen of approximately 64 kD in the human sperm extract. The antibodies showed binding with the live and methanol-fixed human sperm, and significantly (P < 0.001) inhibited human sperm penetration of zona-free hamster oocytes, as well as human sperm binding to human oocyte zona pellucida. These findings indicate that the testis/sperm- specific CV antigen has a role in human sperm function and may find clinical applications in the contraceptive vaccine development and in the specific diagnosis and treatment of male infertility.

Adult↗