Expression profiles of SRY and SOX9 in rabbit gonads: the classical model of mammalian sex differentiation.

Sex determination in mammalian gonads depends on the concerted action of SRY and SOX at the genital ridge. Although most research on the mechanisms involved in sex determination has been done in mice, the study of non-model organisms may indicate the extent of the generalizations currently based on model systems. The present study investigated the correlation between SRY/SOX9 expression patterns and the process of morphogenesis in the developing gonad of the rabbit. In males, the onset of SRY/SOX9 expression closely followed the establishment of undifferentiated genital ridges at 13 to 14 dpc. In contrast to mouse, real-time PCR in the rabbit revealed that levels of SRY/ SOX9 peak after the onset of seminiferous cord formation, while in mouse this occurs before. Furthermore, rabbit gonads maintain low levels of SRY and SOX9 expression in male and female gonads, respectively. In situ hybridization suggests that cells of the mesonephric Bowman capsules, which do not express SRY, may become SOX9-expressing pre-Sertoli cells during the long period of seminiferous cord formation in the rabbit. In contrast to mouse, current results indicate that the patterns of SRY/SOX9 expression associated with the process of gonadal morphogenesis in rabbit appear similar to those of other mammals, including humans.

Coding sequence mutations in the alpha subunit of propionyl-CoA carboxylase in patients with propionic acidemia.

Propionic acidemia is a rare autosomal recessive disorder of intermediary metabolism. It is caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric protein composed of two subunits, alpha and beta. PCC requires ATP and biotin as cofactors for the reaction, the latter enzymatically added onto the alpha subunit. We investigated coding sequence mutations in the alpha subunit of PCC by analyzing fibroblast RNA from propionic acidemia patients deficient in alpha subunit function by single-strand conformation polymorphism and direct sequencing. Five missense mutations and one short in-frame deletion were found among different patients. Four mutations were located in the putative biotin carboxylase domain, whereas the two others were within the 67-amino-acid C-terminal domain previously shown to be required to obtain biotinylation of the alpha subunit. We analyzed fibroblast extracts for the presence of a biotinylated alpha subunit by Western blot analysis using streptavidin coupled to alkaline phosphatase. Four of five cell lines failed to show a biotinylated alpha subunit, regardless of the position of the mutations within the coding sequence. Two mutations located in the biotinylation domain were expressed in an Escherichia coli-based system and shown to abolish biotinylation of the domain. The results suggest that most mutations have a severe impact on the stability or the functionality of the alpha subunit.

Structure of Pit-1 POU domain bound to DNA as a dimer: unexpected arrangement and flexibility.

Pit-1, a member of the POU domain family of transcription factors, characterized by a bipartite DNA-binding domain, serves critical developmental functions based on binding to diverse DNA elements in its target genes. Here we report a high resolution X-ray analysis of the Pit-1 POU domain bound to a DNA element as a homodimer. This analysis reveals that Pit-1 subdomains bind to perpendicular faces of the DNA, rather than opposite faces of the DNA as in Oct-1. This is accomplished by different spacing and orientation of the POU-specific domain. Contrary to previous predictions, the dimerization interface involves the carboxyl terminus of the DNA recognition helix of the homeodomain, which in an extended conformation interacts with specific residues at the amino terminus of helix alpha1 and in the loop between helices alpha3 and alpha4 of the POU-specific domain of the symmetry related monomer. These features suggest the molecular basis of disease-causing mutations in Pit-1 and provide potential basis for the flexible allostery between protein domains and DNA sites in the activation of target genes.

Clustering of mutations in the biotin-binding region of holocarboxylase synthetase in biotin-responsive multiple carboxylase deficiency.

Holocarboxylase synthetase (HCS) catalyses the biotinylation of the four biotin-dependent carboxylases found in humans. A deficiency in HCS results in biotin-responsive multiple carboxylase deficiency (MCD). We have identified six different point mutations in the HCS gene in nine patients with MCD. Two of the mutations are frequent among the MCD patients analyzed. Four of the mutations cluster in the putative biotin-binding domain as deduced from the corresponding Escherichia coli enzyme and consistent with an explanation for biotin-responsiveness based on altered affinity for biotin. The two others may define an additional domain involved in biotin-binding or biotin-mediated stabilization of the protein.

Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli.

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the four biotin-dependent carboxylases in human cells. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS is targeted to the mitochondria and cytoplasm. We isolated 21 human HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complementation of an Escherichia coli birA mutant defective in biotin ligase. Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragment of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid. The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759. Northern blot analysis revealed the presence of a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of poly(A)+ RNA in human tissues. Human HCS shows specific regions of homology with the BirA protein of E. coli and the presumptive biotin ligase of Paracoccus denitrificans. Several forms of HCS mRNA are generated by alternative splicing, and as a result, two mRNA molecules bear different putative translation initiation sites. A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG codon to direct its translation. We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitochondria remain to be confirmed.

Sequence requirements for the biotinylation of carboxyl-terminal fragments of human propionyl-CoA carboxylase alpha subunit expressed in Escherichia coli.

Biotin-dependent enzymes play an essential role in the metabolism of all organisms. Their biotinylation is catalyzed by holoenzyme synthetases, which attach a biotin molecule to a specific lysine residue on the apoenzymes. The sequence flanking the biotin binding site is highly conserved among biotin-dependent enzymes. This sequence conservation might be related to the extensive cross-species activity showed by holoenzyme synthetases. In this study, we have expressed carboxyl-terminal fragments of the alpha subunit of human propionyl-CoA carboxylase (PCC-alpha) in Escherichia coli and used site-directed mutagenesis to determine the sequence requirements for biotinylation by the bacterial holoenzyme synthetase. We show that the carboxyl-terminal 67 amino acids of PCC-alpha act as an independent domain in the biotinylation reaction. Mutations that affect several conserved Gly residues and a Pro-Met-Pro sequence near the biotin binding site are critical for biotinylation. Substitution of the amino acids that flank the biotin acceptor Lys residue or elimination of the last 3 amino acids of the PCC-alpha peptides had little or no effect on their biotinylation despite their high conservation in biotin enzymes.

Mutations participating in interallelic complementation in propionic acidemia.

Deficiency of propionyl-CoA carboxylase (PCC; alpha 4 beta 4) results in the rare, autosomal recessive disease propionic acidemia. Cell fusion experiments have revealed two complementation groups, pccA and pccB, corresponding to defects of the PCCA (alpha-subunit) and PCCB (beta-subunit) genes, respectively. The pccBCC group includes subgroups, pccB and pccC, which are thought to reflect interallelic complementation between certain mutations of the PCCB gene. In this study, we have identified the mutations in two pccB, one pccC, and two pccBC cell lines and have deduced those alleles participating in interallelic complementation. One pccB line was a compound heterozygote of Pro228Leu and Asn536Asp. The latter mutation was also detected in a noncomplementing pccBC line. This leaves Pro228Leu responsible for complementation in the pccB cells. The second pccB line contained an insertional duplication, dupKICK140-143, and a splice mutation IVS + 1 G-->T, located after Lys466. We suggest that the dupKICK mutation is the complementing allele, since the second allele is incompatible with normal splicing. The pccC line studied was homozygous for Arg410Trp, which is necessarily the complementing allele in that line. For a second pccC line, we previously had proposed that delta Ile408 was the complementing allele. We now show that its second allele, "Ins.Del," a 14-bp deletion replaced by a 12-bp insertion beginning at codon 407, fails to complement in homozygous form. We conclude that the interallelic complementation results from mutations in domains that can interact between beta-subunits in the PCC heteromer to restore enzymatic function. On the basis of sequence homology with the Propionibacterium shermanii transcarboxylase 12S subunit, we suggest that the pccC domain, defined by Ile408 and Arg410, may involve the propionyl-CoA binding site.

Studies on the mechanism of biotin uptake by brush-border membrane vesicles of hamster enterocytes.

Several studies on biotin intestinal transport in the hamster have shown a biotin-specific carrier, but there are conflicting reports on whether it is transported actively, or by facilitated diffusion and on its Na+ dependence. We have studied it for the first time using brush-border membrane vesicles (BBMV), with concentrations in a more physiological (nanomolar) range and found an overshoot component, evidencing a carrier-mediated active process, driving the vitamin against a concentration gradient. Uptake was substantially reduced when potassium substituted for sodium. When the vesicles were treated with trypsin, Na(+)-dependent uptake was markedly reduced and the overshoot phenomenon was abolished, providing additional evidence for the carrier-mediated transport. The amount of uptake in a K+ gradient was considered due to passive diffusion and was about 30% of that observed in a Na+ gradient. A similar amount was observed when trypsinized vesicles were incubated in this latter gradient. Our results indicate that in the hamster's brush border intestinal epithelium, Na(+)-dependent active transport is the most important component in the intestinal uptake of biotin at nanomolar concentrations.

Association of pancreatic biotinidase activity and intestinal uptake of biotin and biocytin in hamster and rat.

Pancreatic biotinidase activity was higher in hamster than in rat; these results were reversed in plasma. Uptake was studied in everted intestinal rings. Saturation kinetics at 37 degrees C were observed for biotin in hamster and for biocytin in rat, with a Vmax of 1.83 and 1.05 nmol min-1 ml-1 and an apparent Kt of 25.14 and 40.7 microM, respectively. Biotin uptake by hamster intestine was reduced at 4 degrees C and when choline or potassium replaced sodium; it was inhibited by biocytin only at very high concentrations. Biocytin uptake in the rat was small compared to passive diffusion and was not influenced by sodium or temperature; it was not inhibited by biotin. We observed only passive diffusion of biotin in rat and of biocytin in hamster. Our results suggest that protein-bound biotin may be absorbed mainly in its free form in the hamster. In the rat, on the other hand, at least part of the dietary biotin may be absorbed lysine-bound, as biocytin.