Kevin S. Guise

Isolation and characterization of β-act in gene of carp (Cyprinus carpio)

A beta-actin gene of carp (Cyprinus carpio) was isolated from a genomic EMBL3 library. The nucleotide sequence of the gene indicates six exons spanning 3.6 kb. Southern blot hybridization of restriction endonuclease digests of carp genomic DNA indicate that there are two copies of the beta-actin isotype and several other species of actin genes. The transcriptional start site is 85 bp and 24 bp downstream respectively from consensus CCAAT and TATA promoter elements. The organization of the carp beta-actin gene is identical to that of chicken, human, and rat genes in terms of size, exon/intron locations and junctions and in having a translationally silent first exon. The fish gene is 90% and 99% conserved at the nucleotide and amino acid levels, respectively, with land vertebrate beta-actin genes. Northern blot analysis of beta-actin gene expression indicated that the gene is highly expressed in brain, less so in muscle, and much less so in liver cells. The putative beta-actin proximal promoter of carp, identified by the conservation of known actin regulatory sequences, is transcriptionally active in both mammalian and piscine cells.

Molecular analysis and growth evaluation of northern pike (Esox Iucius) microinjected with growth hormone genes

Gross, M.L., Schneider, J.F., Moav, N., Moav, B., Alvarez, C., Myster, S.H., Liu, Z., Hallerman, E.M., Hackett, P.B., Guise, K.S., Faras, A.J. and Kapuscinski, A.R., 1992. Molecular analysis and growth evaluation of northern pike (Esox lucius) microinjected with growth hormone genes. Aquaculture, 103: 253-273. Bovine (bGH) or chinook salmon (Oncorhynchus tshawytscha) growth hormone (csGH) cDNA genes were transferred by microinjection into newly fertilized northern pike (&OX lucks) eggs. Nonlethal screening of fin tissue showed genomic integration of transgenes in 88 of 1398 putative transgenie fish. Expression of bGH transgenes under transcriptional control of the Rous sarcoma virus long terminal repeat was detected in 36 of 1218 putative transgenic fish examined by radioimmunoassay of blood serum, Bovine growth hormone was also detected in mesodermal tissue of fins from microinjected fish using thin slice immunohistochemistry. Southern hybridizations of six tissues from a sample of 40 microinjected individuals revealed a high degree of mosaicism, with 30% of the fish containing detectable transgenic DNA in one or more tissues and only 41% of these containing detectable transgenes in fins. Growth of microinjected fish was quantitatively evaluated in three experiments. Average weight of microinjected fish was greater than that of controls of the same sex in four out of six groups. Significant growth enhancement (PC 0.05) was detected only for microinjected males in one experiment. Comparisons among molecular assays and individual fish growth in the founder generation indicated that the high degree of mosaicism prevented non-lethal indentification of all transgenic individuals and influences detection of growth enhancement.

Transfer of the gene for neomycin resistance into goldfish, Carassius auratus.

Abstract A recombinant DNA construct containing the neo gene controlled by a promoter from the Rous sarcoma virus (RSV) was microinjected into newly fertilized, dechorionated goldfish ( Carassius auratus ) eggs. The neo gene confers resistance to the neomycin analog drug G-418. Results of Southern blot analyses were consistent with incorporation of single or multiple copies of the gene into the genomic DNA of one examined fish. Evidence of neo mRNA in RNA dot-blot analysis indicated that the RSV promoter had initiated transcription within a piscine genome. The utility of the neo gene as a selectable marker for transgenic fish was evaluated by G-418 selection on newly hatched and juvenile fish but proved inconclusive. Reasons for the discrepancy between neo expression and G-418 selection results are discussed.

Importance of the CArG box in regulation of β-actin-encoding genes

Abstract The β-actin-encoding gene ( Act ) in carp is regulated by several cis -acting regulatory elements including the evolutionarily conserved CC(A/T) 6 GG ( CArG box or serum-response element) sequences positioned in the promoter region between the CAAT and TATA boxes and in the first intron. To address the roles of the two CArG boxes on gene expression, we replaced them with linker sequences. The CArG box in the proximal promoter was not required for promoter activity in tissue-cultured cells, but was required in conjunction with a second CArG box in the first intron to give full expression in transgenic embryos. Likewise, the geometry of cis -acting transcriptional elements in the proximal promoter was more important for expression of transgenic constructs in developing embryos than in tissue-cultured fibroblasts. Mobility-shift and exonuclease mapping experiments indicated that the same or similar protein factors bind around the two CArG boxes, suggesting that interactions between the promoter and the first intron are involved in Act regulation.

Isolation and expression in Escherichia coli of a cDNA clone encoding human β-glucuronidase

Abstract Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of β-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280–289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73 %. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropylthio -β-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.

Enzymatic dechorionation of goldfish, walleye, and northern pike eggs

Abstract Removal ofthe chorion from fish eggs facilitates observation ofpiscine embryogenesis and allows efficient microinjection during gene transfer protocols. Several treatments were screened to evaluate their effectiveness in removing the chorion from newly fertilized eggs of goldfish Carassius auratus, walleye Stizostedion vitreum, and northern pike Esox lucius without destroying egg viability. Goldfish eggs dechorionated in 2.5 mg/mL trypsin showed excellent viability. No effective procedure for dechorionation of walleye eggs was found. Northern pike eggs could be dechorionated in 0.6 mg/mL protease type XXV, but mortalities above 50% were commonly observed.

Gene expression promoted by the RSV long terminal repeat element in transgenic goldfish

Abstract Persistence and levels of expression of the chloramphenicol acetyltrans‐ferase (CAT) marker gene under transcriptional regulation by the 5’ long terminal repeat element of the avian Rous sarcoma virus (RSV) were examined in various tissues of transgenic goldfish, Carassius auratus. Evidence of the CAT transgene was observed in 13 of 20 test individuals, with ten individuals being apparent mosaics for the introduced construct. Above‐background acetyltrans‐ferase activity was observed in tissues from 14 individuals, most frequently and at highest levels in muscle. Acetyltransferase activities in muscle tissue of transgenic individuals were as much as fifty times background. Transgene expression, from the RSV promoter, observed in piscine muscle cells paralleled earlier observations of RSV directed gene expression in avian and mammalian systems.

Mitochondrial DNA Variation in Four Minnesota Populations of Lake Whitefish: Utility as Species and Population Markers

Abstract Restriction fragment length polymorphism (RFLP) analysis was performed on the mitochondria) DNA (mtDNA) of 33 lake whitefish Coregonus clupeaformis from four spawning populations in north-central Minnesota. Diagnostic differences between the RFLP patterns of mtDNA from lake whitefish and cisco C. artedi were useful in the identification of fresh fillets and fresh or frozen carcasses. However, mtDNA analysis was of little use for identification of lake whitefish populations because of the low diversity documented among study populations.