Donald H. Whitmore

The arrestin superfamily: cone arrestins are a fourth family

Arrestins constitute a superfamily of regulatory proteins that down‐regulate phosphorylated G‐protein membrane receptors, including rod and cone photoreceptors and adrenergic receptors. The potential role of arrestin in color visual processes led us to identify a cDNA encoding a cone‐like arrestin in Xenopus laevis, the principle amphibian biological model system. Alignment of 18 deduced amino acid sequences of all known arrestins from both invertebrate and vertebrate species reveals five arrestin families. Further analysis identifies 7 variable and 4 conservative arrestin structural motifs that may identify potential functional domains. The adaptive evolutionary relationship of Xenopus cone arrestin to the arrestin gene tree suggests high intrafamily homology and early gene duplication events.

Differential expression of mRNA and protein encoding retinal and pineal S-antigen during the light/dark cycle.

Abstract: S‐Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well‐characterized molecule that participates in light‐induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S‐antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S‐antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7‐kb mRNA for S‐antigen was observed in both the pineal gland and the retina. Retinal S‐antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S‐antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S‐antigen, identified with two S‐antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb CIOC10, was analyzed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS‐PAGE revealed a single 46‐kDa protein in retina. In contrast, two bands of ∼43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S‐antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland‐specific forms of S‐antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue‐specific structural components for S‐antigen in the retina and pineal gland that are not regulated in the same manner.

Gene Amplification Permits Minimally Invasive Analysis of Fish Mitochondrial DNA

Abstract Fish DNA was extracted from epithelial tissue covering a few scales and used as template for the polymerase chain reaction (PCR). Amplified segments of the mitochondrial DNA gene for 12S ribosomal RNA were subjected to restriction enzyme analysis. Restriction fragment length polymorphisms (RFLPs) are described for several fish species. Direct sequencing of amplified segments from largemouth bass Micropterus salmoides and channel catfish Ictalurus punctatus permitted interspecific comparisons and the development of detailed microrestriction maps that reveal diagnostic allelic variations between the two species. The protocol outlined in this report is offered as an alternative to conventional DNA analysis, which usually requires the obligatory sacrifice offish. It may be particularly advantageous when genetic analysis of valuable brood stock or endangered species is necessary because it obviates the need for animal sacrifice.

Identification of sunfish species by muscle protein isoelectric focusing

Muscle protein phenotypes of nine recognized Lepomis species were developed by isoelectric focusing. Four classes were described based on putative parvalbumin patterns. All nine species could be distinguished by their protein phenotypes. Natural hybrids and their parentage were also identified, and the utility of this technique in studying natural sunfish hybridization is discussed.

Isozymal differention between two species of Prosopis

Abstract The patterns of five multilocus isozyme systems were investigated in seed, shoot and cotyledon tissue of two species of mesquite, Prosopis glandulosa var. glandulosa and P. pallida . The isozymes of malate dehydrogenase, peroxidase, esterase, alcohol dehydrogenase and acid phosphatase from each of these tissues were analysed by starch gel electrophoresis and specific histochemical stains. In the case of each enzyme system examined, there were distinctly different isozymes which could be utilized to differentiate between these two species.

Muscle esterases of the mosquitofish, Gambusia affinis

1. Muscle esterases of Gambusia affinis were separable into 14 electrophoretic bands that formed five groups according to differential physical and chemical properties. 2. Nine phenotypic patterns were observed among the populations sampled, based on variation within the EST-3 group of carboxylesterases. 3. A model for the molecular and genetic bases of the EST-3 polymorphism was proposed on the basis of phenotypic patterns and physicochemical characterization. 4. The isozymes of EST-3 are inferred to be dimeric molecules on the basis of electrophoretic patterns observed following incubation of the EST-3 enzymes with neuraminidase. 5. The binding of sialic acid is believed to be an epigenetic mechanism which operates to maintain the structural integrity of these enzymes.

Parvalbumins for calibrating acidic isoelectric focusing gels

Partially purified fish and amphibian parvalbumins are compared to several proteins commonly used in commercial standard mixtures for calibrating isoelectric focusing gels. Parvalbumins are proffered as useful standards for acidic ranges on the basis of conformity to a set of five criteria.

In vitro pesticide inhibition of muscle esterases of the mosquitofish, Gambusia affinis

Abstract 1. The in vitro inhibition of Gambusia affinis muscle esterases by various pesticides was investigated. 2. The oxygen analogs of several organophosphates were found to be potent inhibitors of muscle esterases. 3. Analytically pure malathion did not inhibit esterases unless preincubated with muscle extracts prior to electrophoresis. 4. It is suggested that extracts of muscle have some capability of malathion degradation.

The molecular and genetic basis for lactate dehydrogenase of the mosquitofish, Gambusia affinis

Abstract Electrophoretic and immunochemical analysis of the lactate dehydrogenase isozyme patterns of several tissues of Gambusia affinis indicated that these isozymic patterns are the products of three LDH loci—A, B and C. 1. 2. The LDH-C locus showed expression restricted to neural and retinal tissue. 2. 3. The LDH-A locus was expressed principally in skeletal muscle. 3. 4. No heterozygosity of these three LDH loci was observed in this investigation.