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.     

Appearanc of cytotoxic cells within the bronchus after local infection with herpes simplex virus.

Cells from rabbit spleens, bronchial washings (BW) and bronchus-associated lymphoid tissues (BALT) were examined for their ability to lyse cells infected with herpes simplex virus (HSV). Specific lysis of HSV-infected cells was mediated by BW cells as early as 4 days after intratracheal infection of the rabbits with the virus whereas lysis by spleen cells and BALT cells was not detected until 7 or more days after infection. Lysis by spleen cells was initially detected 7 days after intraperitoneal injection of the virus but lysis by BW and BALT cells was not observed until 14 days after infection. Although spleen, BW, and BALT cells could lyse antibody-coated target cells, antibodies detectable by antibody-dependent cellular cytotoxicity could not be detected in bronchial washings until 7 or more days after infection. The data suggest that cells capable of direct cytotoxicity of virus-infected cells appear within the bronchus after local infection by the virus.     

Selective assay for herpes simplex viruses expressing thymidine kinase.

A technique for selecting herpes simplex viruses expressing the viral thymidine kinase (TK+) from a population of predominantly TK- viruses was developed. This was accomplished by infecting TK- cells and incubating the cultures under a liquid overlay medium containing methotrexate. Since the TK- cells survive in this medium for only a limited period of time, it was necessary to add fresh uninfected TK- cells 48 h after infection. The technique allowed the detection and quantitation of the TK+ virus fraction in mixtures of TK+ and TK- viruses where the TK+ fraction was present in frequencies as low as 10(-5). It was also used to estimate reversion frequencies and to obtain and analyze TK+ revertants from TK- mutant strains of herpes simplex virus type 1.     

Glycoproteins of herpes simplex virus type 2 as defined by monoclonal antibodies.

We used monoclonal antibodies reacting with glycoproteins specified by herpes simplex virus type 2 (HSV-2) to characterize the individual antigens in terms of structure, processing, and kinetics of synthesis in BHK or Vero infected cells. Our results provided a direct demonstration of the structural identity of the gA and gB proteins of HSV-2 as well as confirmation of the existence of type-specific and type-common domains within the gD molecule. They also show that, with the exception of gC, processing of the viral glycoproteins differs to some extent in Vero and BHK infected cells, possibly as a result of different efficiency of glycosylation or different processing of underglycosylated and unglycosylated products in the two cell types. Finally, we showed that individual HSV-2 glycoproteins are synthesized at greatly different times during the infectious cycle, possibly in response to their different roles in virus replication and assembly.     

Characterization of polypeptides immunoprecipitable from Pichinde virus-infected BHK-21 cells

Using hamster anti-Pichinde virus serum, we immunoprecipitated polypeptides from BHK-21 cells infected with Pichinde virus. Seven immunoprecipitable polypeptides exhibited a time- and multiplicity of infection-dependent appearance when the cultures were pulse-labeled with L-[35S]methionine for 1 h. The predominant polypeptide was a nucleoprotein (NP) of 64,000 daltons. Components of 48,000, 38,000, and 28,000 daltons, when analyzed by two-dimensional tryptic peptide mapping, were found to be derived from NP. After a 3-h chase period, polypeptides of 17,000, 16,500, and 14,000 daltons were evident, and peptide mapping revealed that these three polypeptides were also related to NP. During a series of pulse-chase experiments, a 79,000-dalton glycoprotein (GPC) was cleaved to glycoproteins of 52,000 and 36,000 daltons. Radiolabel in a polypeptide of approximately 200,000 daltons (L) did not chase into smaller cleavage products. L, GPC, and NP were found to be unique by two-dimensional tryptic peptide mapping. Comparison of polypeptides immunoprecipitated from infected cells with structural components of purified virus revealed that L protein was evident in both. This is the first report of a high-molecular-weight polypeptide in Pichinde virus particles and infected cells.     

Enzymatic synthesis of [6-14C]orotidine 5'-monophosphate and its use in the assay of orotate phosphoribosyltransferase and orotidylate decarboxylase

A rapid and efficient method is described for the synthesis of [6-¹⁴C]orotidine 5′-monophosphate from radioactive orotic acid using purified yeast orotate phosphoribosyltransferase and inorganic pyrophosphatase. Radioactive orotidine 5′-monophosphate is purified by ion exchange chromatography and employed in small scale assays of Drosophila orotate phosphoribosyltransferase and orotldylate decarboxylase in which both enzyme activities are simultaneously measured in single reaction mixtures. Radioactive substrate and products are separated for counting using DEAE-cellulose paper chromatograms developed in one or two solvents.