Genomic structure and transcript variants of the bovine DAZL gene.

The Deleted in AZoospermia Like (DAZL) gene is a member of the DAZ family and encodes an RNA-binding protein that is expressed in prenatal and postnatal germ cells of males and females. In the human, there are five highly-related members in the DAZ family, four (DAZ1-4) on the Y chromosome and one (DAZL) on an autosome (HSA3). Mutations in these genes have been linked to severe spermatogenic failure and infertility in men. In the present study, we have cloned and characterized the bovine DAZL (bDAZL) gene. The full-length bDAZL cDNA is predicted to encode a protein of 295 amino acids with an RNA recognition motif. The deduced protein sequence of bDAZL is 96 and 97% similar to human and mouse DAZL, respectively. Fluorescence in situ hybridization (FISH) maps bDAZL to the distal region on BTA1q. The bDAZL gene consists of 11 exons and 10 introns. A bDAZL pseudogene was identified on BTA16. Expression analysis of bDAZL in 13 different tissues by RT-PCR shows that two transcripts, variant 1 (2,996 bp) and variant 2 (1,373 bp), of the bDAZL gene are detected only in testis mRNA. The variants probably result from alternative RNA splicing as variant 1 contains an additional 1,623-bp insertion in the 3' UTR. Our results lay the groundwork for possible single nucleotide polymorphism (SNP) and functional studies of the DAZL gene in cattle.

Influence of excision of a methylene group from Glu-376 (Glu376-->Asp mutation) in the medium chain acyl-CoA dehydrogenase-catalyzed reaction.

The human liver medium chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction proceeds via abstraction of an alpha-proton from the acyl-CoA substrates by the carboxyl group of Glu-376. By using the methods of site-directed mutagenesis, we replaced Glu-376 by Asp (E376D mutation), expressed the wild-type and mutant enzymes in Escherichia coli, purified them to homogeneity, and compared their kinetic properties. The steady-state kinetic data revealed that the E376D mutation impaired (by about 15-20-fold) the turnover rate of the enzyme as well as its inactivation by 2-octynoyl-CoA. There was no selective solvent deuterium isotope effect on enzyme catalysis. These results lead to the suggestion that the carboxyl group of Asp-376 does not serve as efficient catalytic base as the carboxyl group of Glu-376. The E376D mutation impaired the octanoyl-CoA-dependent reductive half-reaction such that the rate-limiting step of enzyme catalysis shifted from the product dissociation step (in the case of the wild-type enzyme) to the flavin reduction step, and abolished the previously noted kinetic and thermodynamic correspondences between the octanoyl-CoA-dependent reductive half-reaction and the enzyme-octenoyl-CoA interaction [Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 33, 8833-8841]. Arguments are presented that the Glu-376-->Asp mutation results in uncoupling between the proton transfer and protein conformational change steps during enzyme catalysis.

The relationship of nitrogen source and in vivo nitrate reductase actiity to root formation in Euphorbia esula cell suspension cultures.

Root formation and in vivo nitrate reductase (NR) activity were determined in leafy spurge cell suspensions. Cells grown in B5 media with 1 mg L(-1) 2,4-D were transferred to B5 media without 2,4-D, but containing either high (92:8) or low (15:85) ratios of nitrogen as NO 3 (-) -N:NH 4 (+) -N. In older cell lines root formation occurred only in the low NO 3 (-) medium with =<30 roots per flask. In younger cell lines root numbers were greatest in the high NO 3 (-) medium (1000 to 3000 per flask). Cells grown in low NO 3 (-) medium were about one-third the final dry weight as those in high NO 3 (-) medium. Root length was consistently greater for cell lines of all ages in the low NO 3 (-) medium. Developmental profiles of NR activity were similar in cell lines of all ages, whether or not roots were formed. NR activity was lower, however, in cultures grown in low NO 3 (-) medium compared to high NO 3 (-) medium. There was no consistent relationship between NR activity and root initiation. Therefore, nitrate reductase does not appear to be a primary target for regulation of leafy spurge growth by chemical application.