Rod opsin cDNA sequence from the sand goby (Pomatoschistus minutus) compared with those of other vertebrates.

The absorbance spectra of rods from the sand goby were measured by using microspectrophotometry. Analysis of the averaged spectra shows that the rod visual pigment has a maximum absorbance (lambda max) at approximately 501 nm. A sand goby retinal cDNA library was constructed and then screened with a partial sand goby rod opsin clone obtained by the polymerase chain reaction (PCR). The screening of the library yielded a full length rod opsin clone. The cDNA sequence and deduced amino acid sequence of this clone are compared with those of other vertebrate rod opsins.     

Recognition domains in assembly of oligomeric membrane proteins

Many integral membrane proteins, particularly receptors on the cell surface, are made up of several polypeptide chains. After translation and insertion into the ER membrane, these subunits must assemble into the mature protein. However, the mechanisms controlling their faithful assembly are largely unknown. Recent evidence has shed some light on two cell surface receptors that use different strategies to assemble their subunits. Zach Hall discusses oligomerization of the T-cell receptor and the acetylcholine receptor.     

The acetycholinesterase gene ofAnopheles stephensi

1. The acetylcholinesterase (AChE) gene from the important malaria vector Anopheles stephensi has been isolated by homology to the Drosophila acetylcholinesterase gene. 2. The complete sequence and intron-exon organization has been determined. The encoded protein has 69% identity to Drosophila AChE and 38 and 36% identity to Torpedo AChE and human butyrylcholinesterase, respectively.     

Adhesion of human dermal reticular fibroblasts on complementary fragments of fibronectin: aging in vivo or in vitro

Attachment, spreading, and microfilament reorganization have been evaluated in human dermal reticular fibroblasts isolated from the inner, upper aspect of the arm of a newborn male (RET5 cells) and a 78-year-old male (RET8 cells). Substrata were tested using a set of complementary fragments from individual polypeptide chains of human plasma fibronectin (pFN) or cellular FNs (cFN). With both cell classes, fragments containing the C-terminal heparin-binding (HepII) domain only elicited linear bundles of microfilaments in spreading cells but no stress fibers; fragments containing the RGDS-dependent cell-binding (CellI) domain elicited only partial spreading with condensations of F-actin at ruffling membranes and at other regions along the plasma membrane. The minimum sequence required to obtain responses identical to those on intact pFN (broad spreading with extensive stress fiber formation) was found in fragment 155 (F155) from the beta chain of pFN; F155 contains both HepII and CellI domains. In contrast, the analogous fragment from the alpha chain of pFN (F145) was notably less effective for generating stress fibers. This evidence along with the better attachment, spreading, and microfilament bundle formation on the HepII fragment from the beta chain than the analogous fragment from the alpha chain indicates that the extra type III homology unit permits more effective interaction of beta chain fragments with cell-surface heparan sulfate proteoglycan and possibly integrin (binding efficiency to the substratum was similar for fragments from both chains). Therefore, alternatively spliced sequences that neighbor binding domains can play significant roles in the interaction of the domain with cell-surface receptors of dermal fibroblasts. Comparison of RET5 responses with those of RET8 cells has identified changes in adhesive mechanisms as cells undergo "aging" processes. Attachment and microfilament bundle formation were far more effective for RET5 cells than for RET8 cells on any of the HepII fragments. Conversely, RET8 cells were far more sensitive to an RGDS-containing peptide in their medium on CellI fragments than RET5 cells. These results together indicate that in vivo aging leads to greater dependence upon cell-surface integrin binding and less dependence upon heparan sulfate proteoglycan binding for responses on FN matrices. When RET5 cells entered senescence (in vitro aging), they also became much more sensitive to peptide A. On several fragments and on intact pFN, RET8 cells generated very thick stress fibers that were observed only on one fragment with RET5 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Dissonance Mutant of Courtship Song in Drosophila Melanogaster: Isolation, Behavior and Cytogenetics

The dissonance mutant of courtship song was induced by chemical mutagenesis. This X-chromosomal mutation causes the D. melanogaster male's acoustical output, resulting from his wing vibrations directed at a female, to include very long and loud tone "pulses." Yet, a given train of pulses starts out as normal, with the signals in all but the shortest singing bouts eventually becoming polycyclic and high-amplitude. The aberrant songs caused by diss (map position, 1-52; cytological interval, 14C1-2 to 14C4-5) were quantitatively compared to those produced by mutant cacophony males, whose pulses are much more uniformly polycyclic (due to a mutation mapping elsewhere on the X chromosome). Males or females expressing diss are normal in several "general" behaviors. Yet diss males not only sing abnormally, but they also exhibit longer-than-normal mating latencies in their courtship of females. These decrements seem to be associated, at least in part, with visually aberrant behavior of diss flies--measured with regard to male courtship per se, and also in tests of more general visual responses. Such defects were found when testing diss males or females, and the genetic etiology of the visual impairments were provisionally mapped to the same locus to which the song abnormality has been localized. Neurogenetic connections between the control of courtship singing behavior and visual system functions are discussed with respect to the new song mutation (diss) and the older one (cac)--which also turned out to be genetically related to a mutation that causes abnormalities of light-induced behavior and physiology.     

Activation of a cryptic gene by excision of a DNA fragment.

The cryptic bgl operon in Escherichia coli K-12 strain 1011A contains a 1.4-kilobase-pair fragment of foreign DNA within the bglF structural gene. The active allele found in its descendant strain, MK1, required the precise excision of that insertion for its activation. Molecular and genetic approaches have shown that strain 1011A possessed an active (bglR+) rather than a silent wild-type (bglR0) allele of the regulatory region and that this change was caused by a point mutation. Our model for the retention of cryptic genes (B. G. Hall, S. Yokoyama, and D. H. Calhoun, Mol. Biol. Evol. 1:109-124, 1983) suggested that the insertion might have been selected to silence a disadvantageous bglR+ allele. We examined the genealogy of strain MK1 and found that the insertion of foreign DNA was not selected for that reason, since it preceded the change to bglR+. This means that the change to bglR+ was also not selected, since the presence of the insertion would not allow expression of the operon. We have calculated the probability of isolating a bglR+ mutation by chance alone as less than 10(-8). We suggest that mutation rates estimated under the usual conditions of exponential growth may be irrelevant to the frequencies of these events under natural conditions.     

Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.     

Amino acid substitutions in mitochondrial ATPase subunit 9 of Saccharomyces cerevisiae leading to oligomycin or venturicidin resistance

A series of isonuclear oligomycin-resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial oli1 gene has been studied. DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex. A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane. Certain amino acid substitutions also confer cross-resistance to venturicidin: these residues define an inner domain for venturicidin resistance. The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.     

Characterization of the intermediate filament proteins of Murine Mammary Gland epithelial cells : Response to collagen substratum

The insoluble cytoskeletal material remaining after detergent lysis of 'Normal' Murine Mammary Gland (NMuMG) cells, growing on plastic or collagen gel substrata, was analyzed by two-dimensional gel electrophoresis. The identity of the cytoskeletal elements was determined by their solubility properties, electrophoretic separation pattern, and immunoreactivity using monoclonal antibodies against intermediate filament proteins (AIF), keratins (AE1 and AE3) and actin. The electrophoretic pattern of the cytoskeletal elements from the NMuMG cell strain was found to be very similar to that of primary mouse mammary epithelial cells. Both NMuMG and primary mammary epithelial cells when grown on collagen exhibited an increased expression of a 49 kD protein with a pI of 5.6, that appeared to be a cytokeratin. Many of the cytoskeletal proteins remained tightly attached to the collagen gel substratum after cell lysis. These results demonstrate that the NMuMG cell strain has retained a stable expression of cytokeratins that remains responsive to the presence of extracellular matrix material.     

Purification to homogeneity of aromatase from human placenta

Aromatase cytochrome P-450 has been purified from human placenta to homogeneity, as demonstrated by electrophoresis on polyacrylamide gels with SDS, and by double diffusion against an antibody raised in rabbits. The enzyme converts androstenedione to estrone (Vmax 13.3 n moles/min/n mole P-450; Km 30 microM) and testosterone to estradiol. Aromatase activity requires P-450, P-450 reductase and NADPH. Enzyme activity is inhibited by anti-aromatase antibodies and by 4-hydroxyandrostenedione. The enzyme shows a molecular weight of 55,000, is extremely unstable and spontaneously forms P-420 with a half-life of 2.5 days.