RESUMEN
The t complex responder (Tcr) encoded by the mouse t haplotype is able to cause phenotypic differences between t and + sperm derived from t/+ males, leading to non-Mendelian inheritance. This capability of Tcr contradicts the concept of phenotypic equivalence proposed for sperm cells, which develop in a syncytium and actively share gene products. By analyzing a Tcr minigene in hemizygous transgenic mice, we show that Tcr gene products are post-meiotically expressed and are retained in the haploid sperm cells. The wild-type allele of Tcr, sperm motility kinase-1 (Smok1), behaves in the same manner, suggesting that Tcr/Smok reveal a common mechanism prone to evolve non-Mendelian inheritance in mammals.
Asunto(s)
Regulación de la Expresión Génica , Patrón de Herencia/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Espermátides/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Fenotipo , Espermátides/ultraestructuraRESUMEN
How restriction enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II restriction endonucleases, namely SsoII (downward arrow CCNGG), PspGI (downward arrow CCWGG), Eco-RII (downward arrow CCWGG), NgoMIV (G downward arrow CCGGC), and Cfr10I (R downward arrow CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of approximately 70 amino acid residues with MboI, a Type II restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913-929). Nevertheless, MboI has a dissimilar DNA specificity (downward arrow GATC) compared with these enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of approximately 70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II restriction enzymes of widely different specificities.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/química , Secuencia de Aminoácidos , Dominio Catalítico , Cromatografía en Gel , Biología Computacional , Reactivos de Enlaces Cruzados/farmacología , ADN/química , ADN/metabolismo , Análisis Mutacional de ADN , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Dimerización , Escherichia coli/metabolismo , Evolución Molecular , Luz , Magnesio/química , Manganeso/química , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Filogenia , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Sales (Química)/farmacología , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Factores de TiempoRESUMEN
We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.