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.      

Exclusion mapping of the X-linked dominant chondrodysplasia punctata/ichthyosis/cataract/short stature (Happle) syndrome: possible involvement of an unstable pre-mutation

Summary Homology with the mouse bare patches mutant suggests that the gene for the X-linked dominant chondrodysplasia punctata / ichthyosis / cataract / short stature syndrome (Happle syndrome) is located in the human Xq28 region. To test this hypothesis, we performed a linkage study in three families comprising a total of 12 informative meioses. Multiple recombinations appear to exclude the Xq28 region as the site of the gene. Surprisingly, multiple crossovers were also found with 26 other markers spread along the rest of the X chromosome. Two-point linkage analysis and analysis of recombination chromosomes seem to exclude the gene from the entire X chromosome. Three different mechanisms are discussed that could explain the apparent exclusion of an X-linked gene from the X chromosome by linkage analysis: (a) different mutations on the X chromosome disturbing X inactivation, (b) metabolic interference, i.e. allele incompatibility of an X-linked gene, and (c) an unstable pre-mutation that can become silent in males. We favour the last explanation, as it would account for the unexpected sex ratio (MF) of 1.21 among surviving siblings, and for the striking clinical variability of the phenotype, including stepwise increases in disease expression in successive generations.     

Mechanisms of protection against herpes simplex virus type 1-induced retinal necrosis by in vitro-activated T lymphocytes.

In BALB/c mice, acute retinal necrosis occurs in the uninoculated eye 8 to 10 days following uniocular anterior chamber inoculation of the KOS strain of herpes simplex virus type 1 (HSV-1). Retinitis in the uninjected eye can be prevented if HSV-1-specific immune effector cells that have been restimulated with virus in vitro are administered intravenously within 1 day of anterior chamber inoculation of virus. We explored further the mechanism of protection afforded by these activated immune effector cells. The results of our studies revealed that optimal protection from retinitis required in vitro restimulation, since infusion of 50 x 10(6) HSV-1-primed but nonrestimulated cells could not protect as well as 10 x 10(6) activated cells. Analysis of both restimulated and nonrestimulated cells showed that only in vitro-restimulated cells were cytotoxic to HSV-1-infected syngeneic target cells. From these studies, we concluded that the ability to kill virus-infected target cells contributed to optimal protection achieved by intravenous administration of activated immune effector cells. Furthermore, T-cell subset depletion of activated immune effector cells demonstrated that both L3T4+ and Lyt-2+ T cells in the transfer inoculum contributed to protection. Additional studies revealed that although the transferred immune effector cells reached the injected eye within 24 h, virus replication in the injected eye was not affected. In the uninjected eye, virus titers were low, consistent with protection of this eye from retinitis. Taken together, the virus recovery results suggest that the interaction of virus with intravenously administered HSV-1-specific immune effector cells which limits virus spread and/or replication of virus probably occurred within the central nervous system and prevented the second wave of virus from entering the uninoculated eye.     

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.     

Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii

Candida yeasts rapidly form peroxisomes of simple function and composition when grown on methanol. Because the induction is both massive and rapid, this system may be useful for a detailed dissection of peroxisomal biogenesis. We report procedures to express peroxisomal proteins in cells and spheroplasts of Candida boidinii to stabilize peroxisomes in a lysate of spheroplasts and to obtain an enriched peroxisomal fraction. With these techniques we have been able to study the assembly of alcohol oxidase, a homo-octomeric flavoprotein, into the organelle in vivo. The primary translation product of alcohol oxidase comigrates on sodium dodecyl sulfate-polyacrylamide gels with the mature subunit. Pulse-chase experiments indicate that the newly synthesized monomer of alcohol oxidase has a half-life of about 20 min in intact cells and 13 min in spheroplasts before conversion to octomer. The monomer first appears in a high speed supernatant of a lysate of spheroplasts and then chases into a purified peroxisomal fraction before or during its octomerization. There is no detectable intermediary organelle involved in this process.     

Mechanism of Polykaryocytosis Associated with Noncytopathic Infection by Measles Virus.

Atherton, John G. (University of Southern California, Los Angeles), Sotiros G. Chaparas, Martha Cremer, and Irving Gordon. Mechanism of polykaryocytosis associated with noncytopathic infection by measles virus. J. Bacteriol. 90:213-219. 1965.-Infection with a measles virus variant resulted not only in formation of polykaryocytes (PK) but also in formation of multicellular immunofluorescent foci (IFF) in which no cytopathic effect could be detected. The ratio of IFF to PK changed from 27 to 4 during the first passage and remained 4 after a second passage. PK were plaques. Plaque assay was linear in the presence of IFF. To investigate the mechanism of PK formation, radioautography was done on cells pulse-labeled with tritiated thymidine before virus multiplication began. The results showed that PK were formed by fusion; there were no PK whose nuclei contained no label, and the proportion of labeled nuclei (32%) and distribution of grain counts was the same in PK as in uninvolved cells, ruling out nuclear replication without concomitant cytoplasmic membrane formation as the mechanism of formation of these PK. Early in PK development, neutral red uptake was markedly increased ("red" plaques). As PK matured, hyperchromicity disappeared ("white" plaques). This sequence provided an index of rate of evolution of PK. Rate of PK maturation was more rapid at 37 than at 32 C.     

A comparison of sperm morphology and silver nitrate staining characteristics in the domestic ferret and the black-footed ferret

Ejaculated sperm from the domestic ferret (Mustela putorius furo) and the black-footed ferret (Mustela nigripes) were compared for differences in morphological abnormalities and argentophilic protein distribution. Thawed domestic ferret sperm was also compared to fresh sperm to determine whether there were any effects on cell morphology due to cryopreservation. There were statistically significant differences between the two species of ferret in two of the categories scored. The domestic ferret had a higher frequency of cells that were bent in the midpiece and in the principal piece, and a higher frequency of headless and tailless cells when compared to the black-footed ferret. There were no statistically significant differences in cell morphology between the fresh and cryopreserved ejaculates of the domestic ferret employing a standard egg yolk cryoextender. Silver nitrate staining distribution was different between the two species in both the head and tail region.     

Internal protein sequence analysis: enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes.

A procedure for the generation and isolation of internal peptide fragments for less than 10 micrograms of protein bound to either polyvinylidene difluoride (PVDF) or nitrocellulose membranes after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) is presented. This technique has produced internal sequence data for 120 peptides, with an average initial yield of 20 pmol. Membrane-bound proteins were enzymatically digested with either trypsin or endoproteinase Lys-C in the presence of 1% hydrogenated Triton X-100/10% acetonitrile/100 mM Tris-HCl, pH 8.0, for 24 h at 37 degrees C. The eluted peptides were then directly isolated by microbore HPLC for subsequent sequence analysis. One percent hydrogenated Triton X-100 did not inhibit enzymatic activity, distort HPLC resolution of peptides, or contain uv-absorbing contaminants that could interfere with peptide identification. Reproducible peptide maps and consistent recoveries are presented for standard proteins (3.5-8.0 micrograms) bound to either membrane, with higher recoveries for PVDF-bound proteins. Ninety percent of the proteins analyzed by this technique have produced results; representative peptide maps and sequence data are presented. This technique has a wide range of applications, particularly for proteins with blocked amino termini or those that can only be purified by SDS-PAGE or 2D isoelectric focusing SDS-PAGE.     

A Free Radical Ubiquitously Associated with Senescence in Plants: Evidence for a Quinone

The interpretation of EPR and ENDOR measurements on an organic free radical which appears to be a universal concomitant of senescence in plants is discussed. On the basis of EPR spectra obtained at 95 GHz it is speculated that the radical is derived from a quinone.