RESUMEN
Protamines and transition proteins (TNP) play a major role during spermatogenesis and spermatid differentiation. By screening a porcine genomic library with the corresponding cDNAs, we isolated the genomic clones for porcine protamines 1 and 2 as well as for transition proteins 1 and 2. Analysis and comparison of exon-intron structure, 5' and 3' untranslated regions and transcription start points revealed features common to their counterparts in other mammals. In contrast to TNP2 the gene for TNP1 is well conserved among species, indicating a non species-specific function in the chromatin condensation of the spermatid nucleus. Furthermore, Southern blot hybridization of digested phage DNA demonstrated close linkage between both protamine genes and the gene for transition protein 2 in a 13-kb stretch of DNA. Our observations support the hypothesis that these genes may have evolved by gene duplication.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Ligamiento Genético , Protaminas/genética , Espermátides/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , ADN , Exones , Intrones , Masculino , Datos de Secuencia Molecular , Porcinos , Transcripción GenéticaRESUMEN
Acrosin is a serine proteinase located in a zymogen form, proacrosin in the acrosome of the sperm. It is released as a consequence of the acrosome reaction and is believed to be the most important enzyme in the fertilization process. In the mouse, the proacrosin gene is transcribed premeiotically in spermatocytes, but protein biosynthesis starts in haploid spermatids and is restricted to the emerging acrosome. Four lines of transgenic mice harboring 2.3 kb of 5' untranslated region of the rat proacrosin gene fused to the CAT-reporter gene were generated by microinjection of fertilized eggs. The chimeric gene was found to be present in 10-100 copies per genome in the different strains. The 5' untranslated region of rat proacrosin gene could properly direct CAT-gene expression to spermatocytes and CAT-mRNA translation to round spermatids as it is known for mouse proacrosin gene. However, CAT protein is not restricted to the acrosome; rather, it is distributed in the spermatid cytoplasm. This could be due to the lack of DNA sequences for a hydrophobic leader peptide that have been found in all mammalian proacrosins studied until now but that was not present in transgene. It can be concluded from our results that cis-acting sequences required for tissue specific proacrosin expression reside on a 2.3-kb restriction fragment and are conserved in the proacrosin genes of mouse and rat.
Asunto(s)
Acrosina/genética , Precursores Enzimáticos/genética , Regulación de la Expresión Génica , Espermatozoides/metabolismo , Acrosina/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Clonación Molecular , ADN , Precursores Enzimáticos/metabolismo , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Ácido Nucleico , Testículo/citología , Testículo/metabolismoRESUMEN
The genes for two protamines (PRM1 and PRM2) and for two transition proteins (TNP1 and TNP2) have been characterized in several mammalian species. In the human, boar, and bull, the genes for PRM1, PRM2, and TNP2 are closely linked over a stretch of DNA 13-15 kb long. Although similar data are not yet available for the mouse and rat, our results suggest that the three genes are similarly linked in these species. The gene for TNP1 in all species studied is located on another chromosome.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 16 , Ligamiento Genético , Genoma , Proteínas Nucleares/genética , Protaminas/genética , Animales , Proteínas Cromosómicas no Histona , Cósmidos , Proteínas de Unión al ADN , Fibroblastos/citología , Fibroblastos/fisiología , Genoma Humano , Humanos , Metafase , Ratones , Ratones EndogámicosRESUMEN
A cDNA clone coding for boar transition protein 2 (TNP 2) was isolated from a randomly primed cDNA library of boar testis. Sequence analysis revealed an open reading frame of 414 bp (corresponding to 138 amino acids), 33 bp of the 5' untranslated and about 300 bp of the 3' untranslated region. As compared to TNP 2 of mouse and rat, similarity with TNP 2 of the boar is approximately 70% at the nucleotide level and only about 40% on the basis of amino acid sequence. The similarity between boar and bull TNP2 is 77% and 64%, respectively. Northern blot experiments with RNA of different boar tissues and in situ hybridization on mature boar testis sections revealed testis-specific expression of the TNP 2 gene which is restricted to haploid germ cells. Hybridization experiments of boar TNP2 cDNA with testicular RNA of boar, bull, rat and mouse revealed decreasing intensities of the hybridization signals. With human testicular RNA no hybridization could be obtained.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , ADN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Bovinos , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sistemas de Lectura Abierta , ARN/metabolismo , Ratas , Alineación de Secuencia , Especificidad de la Especie , PorcinosRESUMEN
Acrosin is a serine proteinase located in the acrosome of the sperm in a zymogen form, proacrosin. As deduced from the cDNA sequences of human, boar, and mouse proacrosin, the enzyme is synthesized as a preproenzyme, preproacrosin, which contains a hydrophobic leader sequence of 15 to 18 amino acid residues. We have isolated the gene coding for mouse proacrosin from a mouse cosmid library, using cDNA clones as probes. The gene comprises six exons, and one of the five introns is located in the 5'-untranslated region. The transcription initiation site of the preproacrosin mRNA could be assigned to the residue T, 581 nucleotides upstream of the translation initiation codon ATG, with primer extension analysis. TATA and CAAT boxes could be identified at positions -26 and -97, respectively. Similar to other serine proteases, the coding sequence encompasses five exons and the three active-site residues His, Asp, and Ser are encoded by three different exons (E2, E3, E5). The proline-rich domain, which is a characteristic feature of the proacrosin polypeptide, is encoded in exon 5 with the serine active-site residue. The gene is located on chromosome 15 of the mouse genome, bands E/F, and is a member of a syntenic group that was mapped on human chromosome 22, q13-qter. During spermatogenesis the proacrosin gene in the mouse is expressed diploid, in contrast to a haploid expression observed in bull, boar, and rat.
Asunto(s)
Acrosina/genética , Precursores Enzimáticos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Bandeo Cromosómico , ADN , Diploidia , Exones , Expresión Génica , Intrones , Masculino , Ratones , Datos de Secuencia Molecular , Mapeo Restrictivo , Espermatocitos/citología , Espermatocitos/metabolismoRESUMEN
The genes for proacrosin, protamines, and transition proteins are exclusively expressed in haploid spermatogenic cells. From the analysis of mouse x rat cell hybrids which segregate rat chromosomes, the rat gene for proacrosin (ACR) was assigned to chromosome 7, that for transition protein 1 (TNP1) to chromosome 9, and the genes for transition protein 2 (TNP2) and protamine 1 (PRM1) to chromosome 10.
Asunto(s)
Acrosina/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas , Precursores Enzimáticos/genética , Protaminas/genética , Animales , Mapeo Cromosómico , ADN/genética , Sondas de ADN , Células Híbridas , Ratones , Hibridación de Ácido Nucleico , RatasRESUMEN
Acrosin is a serine proteinase and located in a zymogen form, proacrosin, in the acrosome of the sperm. As deduced from the cDNA sequences for human and boar proacrosin, the enzyme is synthesized as a preproenzyme, preproacrosin, which contains a hydrophobic leader sequence. Using cDNA clones as probes, we have isolated the gene coding for human proacrosin from a human leucocyte genomic library and a human cosmid library, respectively. The gene contains four introns between 0.2 kb--4.5 kb in length. Similar to other serine proteinases, the coding sequence of the preproacrosin gene is spread over all the five exons of the gene and the three activesite residues His, Asp and Ser are encoded by three different exons. According to the exon-intron structure, preproacrosin is suggested to be closely related to the serine proteinase subfamily containing trypsin and kallikrein. However, the light chain of proacrosin seems to be similar to that of chymotrypsin. The coding of the serine active-site residue together with the proacrosin-specific proline-rich domain in one exon, namely exon E5, let us assume that the nucleotide sequence for the proline-rich domain was generated during evolution by intron-exon transfer from a foreign gene with subsequent intron excision. By primer extension analysis, the transcription initiation site of the preproacrosin mRNA could be assigned to the residue C at -74 nucleotides upstream from the translation initiation codon ATG. In contrast to most other eucaryotic genes, including the known testis-specific genes, typical TATA and CAAT box sequences in convential distances from the 5' end of the transcription start site could not be evaluated in the proacrosin gene.