The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Cellular processing of pre-proparathyroid hormone involves rapid hydrolysis of the leader sequence.

The cellular synthesis of parathyroid hormone (PTH) involves two consecutive cleavages of NH2-terminal peptide sequences from a larger precursor, pre-proparathyroid hormone (Pre-ProPTH). The initial cleavage consists of the removal of an NH2-terminal leader sequence either during or shortly after biosynthesis of the polypeptide chain is complete. To determine the fate of the cleaved leader sequence, we prepared, by chemical synthesis, a peptide based on the known structure of the leader sequence of pre-proparathyroid hormone and used this peptide labeled with 125iodine as a marker to monitor the recovery of the putative cellular leader peptide during extraction and electrophoresis of [35S]methionine-labeled proteins from pulse-labeled parathyroid gland slices. Under conditions in which the recovery of the synthetic leader peptide was 50 to 70%, we found no detectable 35S-labeled product in the region of sodium dodecyl sulfate gels where the synthetic peptide migrates. In view of the known methionine content of pre-proparathyroid hormone and proparathyroid hormone (ProPTH), it would have been possible to detect endogenously labeled leader peptide if present in amounts equal to 0.05% of the amount of labeled ProPTH present in the tissues. These observations indicate that the cellular conversion of Pre-ProPTH to ProPTH involves a rapid hydrolysis of the leader peptide either during or immediately after its removal from the precursor.     

Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ.

Pre-proparathyroid hormone is the major protein synthesized in wheat-germ extracts in response to addition of an 8-15S fraction of parathyroid RNA. The accuracy of the translation of the mRNA from parathyroid tissue was examined by analysis of the carboxyl-terminal tryptic peptide and the amino-terminal amino acid of the protein, by analysis of the size distribution of the mRNA, and by translation of the mRNA in a second cell-free extract. When 8-15S RNA was fractionated on a sucrose gradient containing formamide, RNA that supported the synthesis of pre-proparathyroid hormone was present in a single symmetrical peak, suggesting that it was homogeneous. Analyses by paper chromatography and electrophoresis of the proline-containing tryptic peptides of pre-proparathyroid hormone indicate that they are identical with the corresponding proline-containing peptides of parathyroid hormone. Because the COOH-terminal tryptic peptide of parathyroid hormone contains proline, the data indicate that the COOH termini of pre-proparathyroid hormone and parathyroid hormone are identical. Methionine from initiator [35S]Met-tRNAfMet was rapidly incorporated into pre-proparathyroid hormone by the wheat-germ extract, and a single-step Edman degradation selectively removed almost all of the initiator [35S]methionine present in pre-proparathyroid hormone. Translation of the 8-15S RNA in a cell-free extract from Krebs-II ascites cells resulted in a protein that comigrated with pre-proparathyroid hormone on sodium dodecyl sulfate-acrylamide gel electrophoresis. These data support the conclusion that the wheat-germ system accurately translates the mRNA for parathyroid hormone, and they strengthen the contention that pre-proparathyroid hormone is the initial biosynthetic product.