Peter H. W. Butterworth

Strong expression of foreign genes following direct injection into fish muscle

We report here for the first time direct injection of genes into fish muscle in vivo. Plasmids used contain either SV40 early promoter, rabbit β‐cardiac myosin heavy chain promoter, human MxA promoter of an artificial promoter, fused to a chloramphenicol acetyltransferase (CAT) or β‐galactosidase reporter gene. CAT assays revealed that most gene constructs were highly expressed. Histochemical analysis showed that β‐galactosidase was strongly expressed at the site of injection within muscle fibres. This method provides an excellent system for testing expression of gene constructs, including those of mammalian origin, in fish muscle in vivo and has the potential for fish vaccination.

Purification of the rat liver form B DNA-dependent RNA polymerases

Five differing mammalian DNA-dependent RNA polymerases have been reported [ I-61 . Since the nomenclature of these enzymes is somewhat confused at present, we have adopted here that put forward by Chambon and coworkers [3]. If control of gene expression in animal cells is mediated by DNA-dependent RNA polymerases, the most interesting forms of this enzyme are probably those located in the nucleoplasm of animal cell nuclei. These are forms BI and BII (we can find only trace amounts of AIII) which may be responsible for mRNA and heterogeneous nuclear RNA synthesis. Below, a detailed procedure for the purification of these enzymes from rat liver nuclei is reported. The subunit pattern of the product, as demonstrated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, indicates that it is a mixture of the BI and BII enzymes recently separated by Kedinger, Nuret and Charnbon [31.

A new form of mammalian DNA-dependent RNA polymerase and its relationship to the known forms of the enzyme.

Extraction of mammalian cell nuclei with a buffer of high salt concentration together with sonication yields the form I (a-amanitin resistant) and II (aamanitin sensitive) DNA-dependent RNA polymerase in approximately 1:2 proportions [1 -3 ] . We report here that low salt extraction yields mainly form I polymerase. A low salt (fig. 1) and a high salt (fig. 2) extraction and purification procedure have been devised based on previously reported methodology [ 1 -4 ] . The form I polymerase obtained by low salt extraction can be separated into two species, Ia and Ib, of roughly equal activity by phosphocellulose chromatography. The two forms differ in their relative activities with Mg 2+ and Mn ~÷ ions and on native and denatured DNA. Form Ia is the form I enzyme found previously [1 ,2] . Form Ib is present in only small amounts in unextracted nuclei but this level is enhanced up to almost that of Ia during low salt extraction. The implications of this finding with respect to interconversion between the various polymerase forms are investigated further and a model of the nuclear transcription system presented which explains these and other reported results. 2. Materials and methods

DNA-dependent RNA polymerase activity of Chinese hamster kidney cells sensitive to high concentrations of α-amanitin

Abstract Studies on the DNA-dependent RNA polymerase activities present in Chinese Hamster Kidney Cells have revealed an enzyme inhibited only by high concentrations of α-amanitin that corresponds to the previously described RNA polymerase C. Although primarily isolated from the cytoplasmic fraction derived from these cells, evidence is presented which strongly suggests that RNA polymerase C and the nuclear RNA polymerase III are one and the same enzyme.

A possible novel interaction between the 3′-end of 18 S ribosomal RNA and the 5'-leader sequence of many eukaryotic messenger RNAs

A novel interaction between the 5′‐untranslated region of eukaryotic messenger RNAs and non‐contiguous sequences in the 18 S ribosomal RNA is proposed. The small ribosomal RNA contains, at its 3′‐terminus, a heavily conserved hairpin structure. It is suggested that mRNA 5′‐leader sequence stabilises this structure by interacting with other conserved nucleotides which flank it. Sequences closely related to the required sequence (A—U—C—C—A—C—C) occur quite commonly in eukaryotic mRNAs and are often found immediately upstream from the AUG‐codon. This interaction may have a role in the events which lead up to the initiation of protein synthesis.

The effect of heparin on the structure and template properties of chromatin

The polyanion heparin, by virtue of its charge properties, is able to compete with nucleic acids in many protein-nucleic acid interactions. Thus heparin inhibits both ribonucleases [1-3] and initiation by bacterial [3,4] or eucaryotic DNA-dependent RNA polymerases. The presence of ribonuclease activity in chromatin preparations [3,5] has led to the suggestion that heparin be used to abolish this when studying RNA synthesis from such templates in vitro [3]. How- ever, the present manuscript indicates that heparin also causes gross structural modifications to the chro- matin template.

Structure and methylation patterns of the gene encoding human carbonic anhydrase I.

The gene (CAI) encoding human carbonic anhydrase I (CAI) has been isolated and shown to have a total length of 50 kb. Some 36 kb of this consists of a large intron separating the erythroid-specific promoter from the coding region. A small (54 bp) noncoding exon from within this intron is occasionally found in transcripts. Two different polyadenylation sites have been found, the most distal of which is the most commonly used. Methylation levels near the promoter differ widely in cell lines. In CAI-expressing cells, a region of DNA near the promoter is demethylated in a generally highly methylated background. Surprisingly, non-CAI-expressing cell lines show much lower levels of methylation.

Expression of the human carbonic anhydrase I gene is activated late in fetal erythroid development and regulated by stage-specific trans-acting factors

Summary. Using flow cytometric analysis of red cells from different stages of ontogeny with anti‐CAI antibody, it was shown that the human carbonic anhydrase I (HCAI) gene product appears in a developmental stage‐specific manner. Virtually no CAI protein was detectable in fetal red cells prior to birth. However, at about the time of normal delivery (40 weeks gestation) CAI production is switched on. The proportion of cells containing CAI reaches adult levels during the second half of the first year of life. Northern analysis suggests that the appearance of CAI protein results directly from the activation of the gene and the production of new mRNA. A transient heterokaryon system was set up by fusing the erythroleukaemic cell lines MEL C88 (a mouse cell line in which CAI is expressed) and K562 SAI (a human cell line with an embryonic/fetal phenotype, not expressing CAI). SP6 RNAase mapping of RNA from the fused cells showed activation of the human CAI gene. This shows the developmental stage‐specific expression of HCAI to be regulated by trans‐acting factors.

Assignment of the gene determining human carbonic anhydrase, CAI, to chromosome 8

A cDNA clone complementary to the mRNA encoding the rabbit erythrocyte specific carbonic anhydrase, CAI, has been used as probe for human CAI sequences in the analysis of DNA from panels of rodent/human somatic cell hybrids. The presence of the human CAI gene in all hybrids correlates with the presence of chromosome 8. Together with published mapping data, this assignment indicates that three CA loci are situated on chromosome 8.

Regional localization of carbonic anhydrase genesCA1 andCA3 on human chromosome 8

The human carbonic anhydrase isozymes represent a family of homologous proteins which are important in respiratory function, fluid secretion, and maintenance of cellullar acid-base homeostasis. Using somatic cell genetic techniques we have mapped two of the CA genes (CA1and CA3)to human chromosome 8. In situ hybridization data demonstrates that both CA1and CA3map to the same region (q13–q22) of chromosome 8.