Shirley Gillam

Sequence of the gene for iso-l-cytochrome c in saccharomyces cerevisiae

Abstract The complete sequence of the iso-1-cytochrome c gene of yeast has been determined. The coding region of the gene contains no intervening sequences. The coding strand of the DNA immediately upstream from the coding region contains many fewer G residues than the rest of the coding strand both within and beyond the carboxy terminus of the coding region. One consequence of the reduced number of G residues in this region is the absence of the sequence ATG for 122 nucleotides upstream from the initiating ATG. Together with previous studies on the mRNA and the genetics of yeast iso-1-Cytochrome c, the sequence supports a model in which translation starts at the first AUG down-stream from the 5′ terminus of the mRNA, with no other sequence requirements. It also is evident that iso-1-cytochrome c is synthesized directly and not through an intermediary, longer precursor protein, as is often the case for proteins that interact with membranes. The DNA upstream and downstream from the coding region contains sequences which are potential transcription start and stop signals. The sequence confirms the assignments of non-sense and missense mutations throughout the coding region of the gene and provides a rationale for some mutational characteristics of the gene. Part of the sequence was determined using two new strategies for the Sanger terminator method, both of which obviate the need for restriction fragment isolation and template strand separation.

Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum oligodeoxyribonucleotide length

Abstract A synthetic oligodeoxyribonucleotide mismatched at a single nucleotide to a specific complementary site on wild-type circular oX174 DNA can be used to produce a defined point mutation after in vitro incorporation into closed circular duplex DNA by elongation with DNA polymerase and ligation followed by transfection of Escherichia coli (Hutchison et al., 1978; Gillam et al., 1979). The present study is an investigation of the optimum conditions required for the oligodeoxyribonucleotide-primed reaction for production of transition and transversion mutations in oX174 DNA, using the large (Klenow) fragment of E. coli DNA polymerase I. Under optimum conditions up to 39% of the progeny of transfection are the desired mutant and significant mutation is observed using a heptadeoxyribonucleotide.

Identification and isolation of the yeast cytochrome c gene

The iso-1-cytochrome c gene of yeast has been identified and cloned using a synthetic oligodeoxynucleotide as a hybridization probe. The oligomer d[pT-T-A-G-C-A-G-A-A--C-C-G-G] is complementary to a region near the N terminal coding region of the yeast cyc 1 gene. Of several yeast Eco RI fragments which hybridize to this probe, one is changed in size by a G leads to T mutation which eliminates an Eco RI site within the cyc 1 gene. Both the wild-type and the RI- mutant forms were cloned in lambda gt vectors. Maxam-Gilbert sequencing for 91 nucleotides into the coding region for iso-1-cytochrome c yielded a DNA sequence in perfect correspondence with the known protein sequence.

The isolation and properties of phenylalanine hydroxylase from human liver

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.

Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus

Abstract Current serological assays using whole rubella virus (RV) as a target antigen for detecting RV-specific antibodies fail to define specific RV proteins and antigenic determinants such as hemagglutinin (HA) and virus-neutralizing (VN) epitopes of rubella virus. A panel of El deletion mutants and a subset of E1-specific monoclonal antibodies (MAb) were used for the initial analysis of HA and VN epitopes of E1 glycoprotein. A peptide region (E1193 to E1269.) was found to contain HA and VN epitopes. Using both overlapping synthetic peptides and truncated fusion proteins within this region, the HA epitope defined by MAb 3D9F mapped to amino acid residues El214 to El240, while two VN epitopes defined by MAb 211391-1 and MAb 16A1 OE mapped to amino acid residues El 214 to E1233 and E1219 to E1233, respectively. The epitopes defined in this study are recognized by antibody whether or not the epitopes are glycosylated.

Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: II. In vitro selection of mutant DNA

A method for the in vitro selection of mutant DNA has been devised as an adjunct to the recently developed method for the use of short enzymatically-synthesized oligodeoxyribonucleotides of defined sequence as site-specific mutagens for circular DNA. The selection method uses the mutating oligodeoxyribonucleotide as a primer for Escherichia coli DNA polymerase I (large fragment) under conditions where there is preferential interaction with mutant DNA template. After ligation using T4 DNA ligase, endonuclease S1 is used to degrade single-stranded non-mutant DNA leaving the desired mutant as closed circular duplex DNA. This paper describes the development of the method using mutants in phi X174 DNA as the model system. Studiies on the changes A leads to G and G leads to A at position 587 of phi X174 viral DNA (am3 to wild-type and its reversal) show that one or two cycles of selection can lead to a population of phage consisting of close to 100% mutants.

Nucleotide sequence of the 3′ terminal third of rabbit α-globin messenger RNA: Comparison with human α-globin messenger RNA

Abstract Rabbit and human α-globin complementary DNA was synthesized using the primer (dT 10 )dGdCdC hybridized to globin mRNA with reverse transcriptase, and sequenced using the plus and minus gel sequencing procedure (Sanger and Coulson, 1975; Brownlee and Cartwright, 1977). The complete 3′ noncoding region, 89 nucleotides in length, and one third of the coding region of rabbit α-globin mRNA have thus been sequenced. These data are compared with the near complete 3′ noncoding region sequence of human α-globin mRNA obtained in these and previous studies. The two mRNAs are approximately 80% homologous in their 3′ noncoding regions, except that the human sequence has an insert of 24 nucleotides. A very similar insert in sequence has been described in human β-globin mRNA (Proudfoot, 1977).

Mutational Analysis of the Rubella Virus Nonstructural Polyprotein and Its Cleavage Products in Virus Replication and RNA Synthesis

ABSTRACT Rubella virus nonstructural proteins, translated from input genomic RNA as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. To examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral RNA synthesis, we introduced into a rubella virus infectious cDNA clone a panel of mutations that had variable defective effects on p200 processing. The virus yield and viral RNA synthesis of these mutants were examined. Mutations that completely abolished (C1152S and G1301S) or largely abolished (G1301A) cleavage of p200 resulted in noninfectious virus. Mutations that partially impaired cleavage of p200 (R1299A and G1300A) decreased virus replication. An RNase protection assay revealed that all of the mutants synthesized negative-strand RNA as efficiently as the wild type does but produced lower levels of positive-strand RNA. Our results demonstrated that processing of rubella virus nonstructural protein is crucial for virus replication and that uncleaved p200 could function in negative-strand RNA synthesis, whereas the cleavage products p150 and p90 are required for efficient positive-strand RNA synthesis.

Rubella Virus Nonstructural Protein Protease Domains Involved in trans- and cis-Cleavage Activities

ABSTRACT Rubella virus (RV) genomic RNA contains two large open reading frames (ORFs): a 5′-proximal ORF encoding nonstructural proteins (NSPs) that function primarily in viral RNA replication and a 3′-proximal ORF encoding the viral structural proteins. Proteolytic processing of the RV NSP ORF translation product p200 is essential for viral replication. Processing of p200 to two mature products (p150 and p90) in the order NH2-p150-p90-COOH is carried out by an RV-encoded protease residing in the C-terminal region of p150. The RV nonstructural protease (NS-pro) belongs to a viral papain-like protease family that cleaves the polyprotein both in trans and incis. A conserved X domain of unknown function was found from previous sequence analysis to be associated with NS-pro. To define the domains responsible for cis- andtrans-cleavage activities and the function of the X domain in terms of protease activity, an in vitro translation system was employed. We demonstrated that the NSP region from residue 920 to 1296 is necessary for trans-cleavage activity. The domain from residue 920 to 1020 is not required for cis-cleavage activity. The X domain located between residues 834 and 940, outside the regions responsible for both cis- andtrans-cleavage activities of NS-pro, was found to be important for NS-pro trans-cleavage activity but not forcis-cleavage activity. Analysis of sequence homology and secondary structure of the RV NS-pro catalytic region reveals a folding structure similar to that of papain.

Analysis of rubella virus E1 glycosylation mutants expressed in COS cells

cDNA clones encoding the envelope glycoprotein E1 of rubella virus (RV) were altered by site-directed mutagenesis at consensus sites for addition of N-linked glycans. The resulting plasmids were introduced into COS cells and the mutant E1 proteins were analyzed by indirect immunofluorescence, radioimmunoprecipitation, and immunoblotting. We found that RV E1 contains three N-linked oligosaccharides, each approximately 2 kDa in size. Although lack of glycosylation did not appear to affect targeting of E1 to the Golgi region, mutants lacking N-linked glycans at Asn 177 and Asn 209 failed to bind anti-E1 antibodies under nonreducing conditions. Our results suggest that glycosylation may be important for expression of important immunologic epitopes on RV E1.