Autosomal recessive nonsyndromic neurosensory deafness at DFNB1 not associated with the compound-heterozygous GJB2 (connexin 26) genotype M34T/167delT.

Previous studies of the gap-junction beta-2 subunit gene GJB2 (connexin 26) have suggested that the 101T-->C (M34T) nucleotide substitution may be a mutant allele responsible for recessive deafness DFNB1. This hypothesis was consistent with observations of negligible intercellular coupling and gap-junction assembly of the M34T allele product expressed in Xenopus oocytes and HeLa cells. The results of our current study of a family cosegregating the 167delT allele of GJB2 and severe DFNB1 deafness demonstrate that this phenotype did not cosegregate with the compound-heterozygous genotype M34T/167delT. Since 167delT is a null allele of GJB2, this result indicates that the in vivo activity of a single M34T allele is not sufficiently reduced to cause the typical deafness phenotype associated with DFNB1. This observation raises the possibility that other GJB2 missense substitutions may not be recessive mutations that cause severe deafness and emphasizes the importance of observing cosegregation with deafness in large families to confirm that these missense alleles are mutant DFNB1 alleles.

Completion of the mouse aggrecan gene structure and identification of the defect in the cmd-Bc mouse as a near complete deletion of the murine aggrecan gene.

Mouse cartilage matrix deficiency (cmd), an autosomal recessive phenotype caused by absence of aggrecan, maps to Chromosome (Chr) 7 and is caused by a 7-bp deletion in exon 5 generating a premature stop codon (Watanabe et al. 1994). Another spontaneous mutation with the same locus and phenotype, cmd-Bc, has now been defined as the complete loss of exons 2 to 18, resulting in a significantly shortened mRNA (1.2 kb). The upstream breakpoint is in intron 1, 18. 8 kb 3' of exon 1; the downstream breakpoint lies 10.5 kb past the final aggrecan exon 18. The deletion is flanked by sequences homologous to topoisomerase I and II cleavage sites and a 7-bp direct repeat, suggesting the defect resulted from a nonhomologous recombination event. Additionally, the size of the first intron and the intron-exon structure between exons 12 and 14 were determined, establishing the length of the murine aggrecan gene as 68.6 kb. This report completes the structural analysis of the murine aggrecan gene, defines a second null mutation, and reinforces the importance of aggrecan in development.

Genomic organization of the mouse and human genes encoding the ATP sulfurylase/adenosine 5'-phosphosulfate kinase isoform SK2.

Mammalian ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase consists of kinase and sulfurylase domains, and catalyzes two sequential reactions to synthesize the universal sulfate donor, phosphoadenosine phosphosulfate (PAPS). In simpler organisms, the ATP sulfurylase and APS kinase reactions are catalyzed by separate enzymes encoded by two or three genes, suggesting that a fusion of separate genes during the course of evolution generated the bifunctional enzyme. We have characterized the genomic structure of the PAPS synthetase SK2 isoform genes for mouse (MSK2) and human (HSK2) and analyzed the possible fusion region. The MSK2 and HSK2 genes exhibit a common structure of 13 exons, including a 15-nucleotide alternatively spliced exon 8. Enzyme activities of several bacterially expressed exon assemblages showed exons 1-6 encode APS kinase, while exons 6-13 encode ATP sulfurylase. The MSK2 construct without the exon 6-encoded peptide showed no kinase or sulfurylase activity, demonstrating that exon 6 encodes sequences required for both activities. Exon 1 and its 5'-flanking sequence are highly divergent between the two species, and intron 1 of the HSK2 gene contains a region similar to the MSK2 promoter sequence, suggesting that it may be the remnant of a now-superceded regulatory region. The HSK2 promoter contains a GC-rich region, not present in the mouse promoter, and has few transcription factor binding sites in common with MSK2. These differences in the two promoter regions suggest that species-specific mechanisms regulate expression of the SK2 isoform.

A member of a family of sulfate-activating enzymes causes murine brachymorphism.

Sulfation is critical to the function of a wide variety of biomolecules. This common modification requires the enzymatic synthesis of an activated sulfate donor, phosphoadenosine-phosphosulfate (PAPS). In higher organisms PAPS synthesis is catalyzed by a bifunctional sulfurylase kinase (SK) polypeptide having both ATP-sulfurylase and adenosine-phosphosulfate kinase activities. We report the identification of a gene family encoding murine SK proteins with these two activities. A family member, SK2, colocalizes with the locus for the autosomal recessive murine phenotype brachymorphism. Brachymorphic mice have normal lifespans, but abnormal hepatic detoxification, bleeding times, and postnatal growth, the latter being attributed to undersulfation of cartilage proteoglycan. A missense mutation in the SK2 coding sequence of bm mice that alters a highly conserved amino acid residue destroys adenosine-phosphosulfate kinase activity and therefore the ability of SK2 to synthesize PAPS. We conclude that a family of SK genes are responsible for sulfate activation in mammals, that a mutation in SK2 causes murine brachymorphism, and that members of this gene family have nonredundant, tissue-specific roles.

Synthesis of turkey Pit-1 mRNA variants by alternative splicing and transcription initiation.

The gene encoding turkey Pit-1/GHF-1 (tPit-1) spans approximately 12 kilobases (kb) and consists of 7 exons. One exon, which is located between exons 2 and 3, is designated exon 2a and codes for 38 amino acids not found in mammalian Pit-1. Because all tPit-1 variants contain exon 2a, they are denoted with an asterisk (*) to distinguish them from comparable mammalian Pit-1s. Three tPit-1 variants are generated by alternative splicing and transcription initiation. Splicing of exon 1 to an alternative acceptor splice site in exon 2 results in a 28 amino acid insertion in tPit-1beta* relative to tPit-1*. A transcript unique to the turkey has been identified by RT-PCR and RNase mapping. This transcript, designated tPit-1W*, arises following transcription initiation upstream of the alternative acceptor splice site in exon 2. In turkey pituitary, the mRNA for the tPit-1* variant is the most abundant, the tPit-1W* variant is intermediate, and the tPit-1beta* variant is the least abundant.

The turkey prolactin-encoding gene and its regulatory region.

Overlapping prolactin (Prl) lambda clones were isolated from a turkey genomic library. The 6.7-kb turkey Prl gene consists of five exons. Major transcription start points were located by primer extension 51-53 nucleotides upstream from the Met start codon. No estrogen response element (ERE) was found, but two regions similar to mammalian Pit-1/GHF-1-binding sites were identified by computer analysis. This suggests that transcription of the turkey Prl gene may be regulated by Pit-1/GHF-1, and not by the estrogen receptor.

Photostimulation changes the pattern of luteinizing hormone secretion in turkey hens.

Changes in the secretory pattern of luteinizing hormone (LH) were determined in turkey hens during initiation of reproduction. Photosensitive hens were switched from a short day photoschedule of 6L:18D (light:dark) to 14L:10D and serially bled six times during the next 6 weeks of photostimulation and early after ovulatory cycles had begun. The pattern of LH secretion at 0-1 day of photostimulation was characterized by a low baseline concentration of LH with numerous low frequency, high amplitude pulses. This pattern changed at Days 4-5 and 11-12 of photostimulation (hens photostimulated but not yet laying) to a pattern characterized by a high baseline with few ill defined pulses of very short duration. Once the hens had initiated egg production, ovulatory surges of LH (duration of 358 +/- 104 min) were superimposed on the basic pattern seen during photostimulation. The ascending limb of the ovulatory surge was much shorter in duration than the descending limb. No diurnal variation was detected in baseline concentration before or during photostimulation or after egg production had begun. It was concluded that photosensitive turkey hens secrete LH in low frequency; high amplitude pulses superimposed on a low baseline. During photostimulation, this pattern changes to one characterized by a high baseline with few pulses of low amplitude and short duration. This pattern of secretion is stimulatory to the ovary and continues until egg laying begins when it is modified by ovulatory surges of LH, characterized by short duration ascending limbs and longer duration descending limbs, which are superimposed on the high baseline pattern noted during photostimulation.

Coordinate pattern of secretion of luteinizing hormone and testosterone in mature male turkeys under continuous and intermittent photoschedules.

Secretory patterns of luteinizing hormone (LH) and testosterone (T) were determined in sexually mature male turkeys housed under Continuous [14 h light (L): 10 h dark (D), n = 3)] or Intermittent [8 x (1L:2D), n = 3] photoschedules. Jugular vein cannulas were implanted on Day 0 and serial blood samples were taken at 5-min intervals for 12 h on Day 3 and again 4 to 7 d later. Most increases in T concentration were preceded by an increase in LH concentration for both photoschedules. Treatment differences for overall mean T [1.77 +/- .58 (SE) ng/mL], baseline T (.92 +/- .30 ng/mL), T peak number (6.1 +/- 1.2 per 12 h), T peak amplitude (2.77 +/- 1.70 ng/mL), or T peak length (79 +/- 24 min) were not significant (P > .05). The number of LH peaks was greater in the Continuous than Intermittent group (11.7 +/- .6 vs 6.8 +/- 1.4 per 12 h, P < .05), but there were no treatment differences in overall mean LH (4.56 +/- 2.09 ng/mL), baseline LH (4.10 +/- 2.11 ng/mL), LH peak amplitude (2.80 +/- .92 ng/mL), or LH peak length (15.9 +/- 3.1 min). There were no differences in T or LH concentrations between the photo- and scotophases in the continuous group, and pulses of both were seen in both phases in both lighting treatments. It was concluded that minor differences in patterns of LH secretion may occur between Continuous and Intermittent stimulatory lighting treatments in sexually mature male turkeys.

Effects of glucagon infusion, alone or in combination with somatostatin, on plasma growth hormone and metabolite levels in young female turkeys under different feeding regimens.

The lipolytic effect of glucagon alone or in combination with somatostatin-14 (SRIF-14) was studied in 9- to 11-wk-old female turkeys that either consumed feed ad libitum or were deprived of feed (FD) for 20 h. During the infusion of various doses of glucagon (.005 to 1.5 micrograms glucagon/min), blood samples were serially collected every 10 min for determination of plasma concentrations of growth hormone (GH) and energy-related metabolites [nonesterified fatty acids (NEFA), glucose, and triacylglycerol (TG)]. Glucagon infusion resulted in decreases (P < .05) in plasma GH in both groups of turkeys. During glucagon infusion, dose-dependent increases in both glucose and NEFA occurred, with greater relative increases in the ad libitum turkeys. In contrast, plasma TG was decreased by glucagon infusion in the ad libitum turkeys but unchanged in the FD turkeys. The effect of simultaneous infusion of SRIF-14 and glucagon on GH and NEFA concentrations varied depending on the previous hormonal treatment. Plasma glucose was not affected by SRIF-14 in either group of turkeys. The present studies show that: 1) glucagon is an important agent for directing carbohydrate and lipid metabolism in growing female turkeys; 2) SRIF-14 has little effect on carbohydrate metabolism, but does affect lipolysis in a complex manner with glucagon; and 3) in vivo, glucagon depresses plasma GH.

Effects of somatostatin on plasma growth hormone and metabolite concentrations in fed and feed-deprived young female turkeys.

The sensitivity and magnitude of response to somatostatin-14 (SRIF-14) infusion in terms of changes in plasma concentrations of growth hormone (GH) and energy-related metabolites [nonesterified fatty acids (NEFA), glucose, and triacylglycerides (TG)] were determined in 9- to 11-wk-old fed or feed-deprived (FD; 20 h) female turkeys. The turkeys were sequentially infused with increasing doses of SRIF-14 (.0015 to 1.5 micrograms/min) concurrent with serial collection of blood samples (every 10 min). Plasma GH and NEFA concentrations were lower in fed than in FD birds. In fed birds only NEFA was minimally altered by SRIF-14 infusion. In FD birds, GH was decreased, but only at the highest dose of SRIF-14 (1.5 micrograms/min), whereas NEFA was decreased in a dose-dependent manner from .05 to 1.5 micrograms/min. Very rapid postinfusion rebounds of plasma GH and NEFA were observed in FD birds. Plasma glucose was not influenced by SRIF-14 in either fed or FD birds. Concentrations of plasma TG were depressed slightly by SRIF-14 in FD but not in fed birds. The present studies indicate that 1) FD birds are more sensitive to SRIF-14 infusion than are fed birds; 2) in FD birds, inhibition of lipolysis (indicated by plasma NEFA) is sensitive to lower dosages of SRIF-14 infusion than is inhibition of GH; and 3) SRIF-14 has little if any effect on TG and glucose metabolism.