Newt RAD51: Cloning of cDNA and analysis of gene expression during spermatogenesis

Abstract

Animals and reagentsAdult male newts (C. pyrrhogaster) collected during winter and early spring, were purchased from Hamamatsu Seibutsu Kyozai Ltd, Hamamatsu, Japan. Adult female newts were collected during autumn. All chemicals were obtained from Nacalai, Kyoto, Japan, unless otherwise stated. A cDNA encoding a newt homolog of Escherichia coli RecA and yeast RAD51 from a testis cDNA library was isolated. The newt RAD51 (nRAD51) cDNA predicted a 337 amino acid protein with a 95-96% amino acid identity to Xenopus and mammalian RAD51. Northern blot analysis showed that nRAD51 mRNA, 1.7 kb in length, was expressed strongly in the testis and ovary, but weakly in the liver, kidney and brain. In situ hybridization revealed that expression of nRAD51 mRNA was barely observed in primary spermatogonia (one cell in a cyst) and early secondary spermatogonia (two to four cells in a cyst), but increased in late secondary spermatogonia (≥ eight cells in a cyst), reaching a maximum level in leptotene-zygotene spermatocytes, and thereafter declined. These results suggest that nRAD51 is involved in mitotic recombination in spermatogonia as well as in meiotic recombination in spermatocytes.

Conversion from mitosis to meiosis: Morphology and expression of proliferating cell nuclear antigen (PCNA) and Dmc1 during newt spermatogenesis

“…The presence of PCNA during meiotic prophase suggests that, in addition to replication, PCNA functions in DNA excision repair during homologous recombination ( Wrobel et al 1996 ). This proposal is further supported by the fact that mRNA for RAD51, an E. coli RecA‐like gene involved in homologous recombination and DNA repair during mitosis and meiosis ( Shinohara et al 1992 ), displays a similar pattern of expression in newt ( Yamamoto et al 1999 ) and mouse ( Kamel et al 1997 ) spermatogenic cells.…”

“…In situ hybridization was carried out as described previously ( Yamamoto et al 1999 ). Sense and antisense DIG‐labeled cRNA probes were prepared as described earlier and hydrolyzed in 100 m M Na 2 CO 3 (pH 10.2).…”

Genetic potential for disease resistance in a critically endangered frog decimated by chytridiomycosis

“…In Xenopus, this gene is 521 involved in genetic recombination and DNA repair, and is likely involved in meiotic recombination 522 due to high expression levels in testes and ovaries (Maeshima et al 1996). RAD51 has also been 523 shown to be strongly expressed in newt testes during spermatogenesis (Yamamoto et al 1999), a 524 process that is increased in Bd-infected frogs (Brannelly et al 2016a). Interestingly, another SNP 525 identified under selection in our study had homology to an outer dense fiber that maintains the 526 elastic structure of sperm tails (2794), further highlighting the potential role of increased 527 reproductive effort as a response to Bd infection.…”