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F de Ferra

Publications and source records attributed to F de Ferra.

At least 19 recordsLinked to original sources
PubMed1999

Manipulation of the DNA coding for the desulphurizing activity in a new isolate of Arthrobacter sp.

A new bacterial strain able to cleave C-S bonds from organosulphur heterocyclic compounds through the 4-S pathway and tentatively classified as Arthrobacter sp. was recently isolated. In the present short article we describe the cloning and the characterization of the DNA encoding the enzymes responsible for desulphurization in this microorganism, referred to as Arthrobacter sp. DS7. The desulphurization operon was found to be located in a large plasmid that also bears the genes conferring cadmium and arsenic resistance. By shortening this plasmid, a new cloning vector was prepared and used to obtain a recombinant derivative strain that desulphurizes dibenzothiophene despite of the presence of inorganic sulphur in the growth medium.

Arthrobacter↗
PubMed1997

Engineering of peptide synthetases. Key role of the thioesterase-like domain for efficient production of recombinant peptides.

Peptide synthetases are large enzymatic complexes that catalyze the synthesis of biologically active peptides in microorganisms and fungi and typically have an unusual structure and sequence. Peptide synthetases have recently been engineered to modify the substrate specificity to produce peptides of a new sequence. In this study we show that surfactin synthetase can also be modified by moving the carboxyl-terminal intrinsic thioesterase region to the end of the internal amino acid binding domains, thus generating strains that produce new truncated peptides of the predicted sequence. Omission of the thioesterase domain results in nonproducing strains, thus showing the essential role of this region and the possibility of obtaining peptides of different lengths by genetic engineering. Secretion of the peptides depends on the presence of a functional sfp gene.

Amino Acid Sequence↗
PubMed1994

Characterization of the surfactin synthetase multi-enzyme complex.

Three subunits (srfAORF1, srfAORF2 and srfAORF3) of the Bacillus subtilis surfactin synthetase multi-enzyme complex have been identified by SDS-PAGE and Western blot analyses. In accordance with the sequence analysis of the surfactin (srfA) operon, the protein subunits have a molecular mass of 402,000 Da, 401,000 Da and 144,000 Da, respectively. Confirmation of the identity of the proteins was obtained by analysing the total protein content of a number of mutant strains which harbour deletions or insertions either in the srfA promoter or in different positions within the srfA operon. The three subunits were partially purified by means of a series of chromatographic steps including ion-exchange chromatography, hydrophobic chromatography and gel filtration chromatography. The partially purified proteins were used in activity assays to establish their amino-acid recognition specificity. In agreement with previously published results, this analysis showed that srfAORF1 recognizes glutamic acid and Leu, srfAORF2 recognizes Val, aspartic acid and Leu and srfAORF3 recognizes Leu. In addition, the subunits can activate and bind other amino acids, although with lower specificity. In particular, srfAORF1 binds Val, Ile and aspartic acid, srfAORF2 glutamic acid and Ile and srfAORF3 Ile and Val. Competition experiments as well as sequence comparison strongly suggest that the Leu binding sites of the three subunits can accept, beside Leu, Ile and Val. The kinetic parameters of srfAORF3 for Leu, Ile and Val have been determined.

Amino Acid Sequence↗
PubMed1993

Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis.

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilobases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.

Amino Acid Isomerases↗
PubMed1993

Characterization of the srfA locus of Bacillus subtilis: only the valine-activating domain of srfA is involved in the establishment of genetic competence.

srfA is a locus required for the production of the lipopeptide antibiotic surfactin. This locus is also necessary for efficient sporulation and competence development. Mutations in the 5' portion of the srfA operon affect all three of these processes, whereas mutations in the 3' portion of srfA only affect sporulation and surfactin production. Analysis of the proteins encoded by the srfA locus revealed seven large domains which are likely to be responsible for the activation and binding of the seven amino acids of surfactin. Identification of the amino acid that is activated by the srfA domains was determined by amino acid-dependent pyrophosphate exchange reactions on partially purified cell extracts of strains carrying different srfA mutations. These results indicate colinearity between the order of the domains in the srfA locus and the amino acid sequence of surfactin. The minimal genetic element of srfA required for the establishment of competence was shown to be the 5' region of the second open reading of srfA, which encodes the valine activation domain. This portion of srfA, when cloned on a plasmid, complemented the competence deficiency of a srfA deletion mutant in trans.

Amino Acid Sequence↗
PubMed1991

Prochymosin expression in Bacillus subtilis.

Prochymosin (PC) sequence was cloned in Bacillus subtilis using two kinds of plasmid constructions. In plasmid pSM316 the cDNA was inserted to obtain the intracellular expression of the enzyme. The enzyme turned out to be expressed in an insoluble form which could be converted to native enzyme under proper denaturing and refolding conditions. The levels of intracellular expression of PC were further enhanced by modifying the 5' region of the gene in a way that a two-cistron expression system was created. For the PC secretion, the cDNA was fused to the subtilisin leader sequence and expressed under the control of the B. subtilis neutral protease promoter. A properly folded PC was secreted by the cells, although to low levels.

Amino Acid Sequence↗
PubMed1991

A new human growth hormone production process using a recombinant Bacillus subtilis strain.

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.

Amino Acid Sequence↗
PubMed1991

Expression of Bordetella pertussis fimbrial (fim) genes in Bordetella bronchiseptica: fimX is expressed at a low level and vir-regulated.

To identify the elements involved in Bordetella pertussis fimbriae regulation and to determine whether fimX is an expressed gene, the promoter regions of fimX, fim2 and fim3 from strain BPSA1 were isolated and linked to the promoterless CAT gene in pLAFR2. By following CAT activity in Bordetella bronchiseptica vir+ and vir- strains we established that the fimX promoter, like those for fim2 and fim3, is active, although at a low level, and vir-regulated. This suggests that the fimX protein might be produced in minute quantities which are not detectable by conventional methods. Comparison of the three fim promoter sequences and transcriptional activities identifies two conserved elements necessary for transcription in the -60 to -20 region: the 'fim box' and the 'C-stretch'. Mutations in these two sequences drastically reduce transcription and alter the interaction with vir components, suggesting a role for the two elements in the regulation of fim genes. Finally, we suggest that the apparent constitutive nature of fim3 in BPSA1 is due to a modification in the length of the 'C-stretch'.

Base Sequence↗
PubMed1990

Differential exon expression in myelin basic protein transcripts during central nervous system (CNS) remyelination.

1. In order to characterize some of the molecular events leading to repair of myelin in the adult central nervous system (CNS), we examined the expression of transcripts for myelin basic protein (MBP) during remyelination in the mouse. C57B1/6 mice develop a demyelinating disease when glial cells are selectively infected by the A59 strain of mouse coronavirus. The virus is spontaneously cleared from the mice by 4 weeks postinfection (WPI), a time when remyelination is starting. 2. At 3 WPI total MBP transcripts are decreased by 75% in demyelinating lesions compared to control white matter. Using RNase protection assays and in situ hybridization with probes for particular MBP exons, we detected an increase in MBP transcripts containing exon 2 information, coincident with the earliest histological signs of remyelination. 3. The expression of MBP transcripts containing exon 2 information was first seen clustered in the perinuclear cytoplasm of oligodendrocytes scattered within the lesions. This is reminiscent of the increased levels and perinuclear clustering of MBP transcripts containing exon 2 seen during early developmental myelination. The peak abundance of exon 2-containing transcripts in the lesions was 13-fold that seen in control white matter. At later stages of remyelination, additional forms of MBP transcripts (without exon 2) increased and their distribution was more diffuse. 4. Thus, during remyelination, preforms of MBP transcripts, which are normally present at low levels in the adult CNS, are abundantly expressed and regulated in a manner similar to that observed in developmental myelination.

Animals↗
PubMed1988

A rapid and versatile site-directed method of mutagenesis for double-stranded plasmid DNA.

This paper describes a new method for site-directed mutagenesis which allows mutations by deletion, insertion or substitution of large fragments of DNA with more than 50% efficiency and does not require subcloning in a single-stranded (ss) DNA vehicle. The site of mutagenesis is removed from a linearized plasmid DNA by BAL 31 digestion, ss DNA regions are generated by limited exonuclease treatment and the mutated target site is reconstituted by annealing of the plasmid DNA to a 35-70 nucleotide long mutated ss oligodeoxynucleotide containing the desired mutation. The circularized plasmid is finally used to transform directly Escherichia coli competent cells.

Base Sequence↗
PubMed1988

Cloning of a novel pilin-like gene from Bordetella pertussis: homology to the fim2 gene.

A search for pilin genes in a Bordetella pertussis (Bp) genomic library has led to the identification of several clones which hybridize to synthetic oligonucleotides with sequences derived from amino acid sequences of Bp fimbrial subunits. One of these clones (corresponding to a gene we have named fimX) contains an open reading frame encoding a protein with a molecular weight of about 20 kD and a sequence similar but not identical to the fimbrial subunit fim2 and to other fimbrial protein sequences. In this communication we present the cloning and nucleotide sequence of the fimX gene and its homology to the fim2 gene. A genomic analysis on the positional relationship between the two genes is also presented.

Amino Acid Sequence↗
PubMed1988

Bordetella pertussis pilin and pilin-like genes.

Bordetella pertussis fimbriae, which mediate the adherence of the microorganism to the epithelium of the respiratory tract, elicit a protective immunogenic response which could be taken advantage of for the creation of an acellular vaccine against pertussis. A few of the fimbrial proteins have been partially characterized by immunological means which recently the cloning and characterization of pilin and pilin-like genes are allowing us to study their structure, regulation of expression during the infectious cycle of Bordetella pertussis, and the basis for antigenic variation. In this communication we will review recent data on Bordetella pertussis pilin and pilin-like genes and their products, with particular emphasis on data most relevant to the design of an acellular vaccine.

Amino Acid Sequence↗
PubMed1986

Identification of three forms of human myelin basic protein by cDNA cloning.

We have isolated cDNA clones encoding three separate forms of human myelin basic protein (MBP), 21.5, 18.5, and 17.2 kDa, and have determined the nucleotide sequence of each. The three forms share a common sequence but differ by the inclusion of a 26-residue amino acid sequence near the N terminus of the 21.5-kDa protein or by the absence of an 11-residue amino acid sequence near the C terminus of the 17.2-kDa protein. The sequences either added to or deleted from the major 18.5-kDa MBP correspond exactly to exons 2 and 5 of the mouse MBP gene, suggesting that the human and mouse genes have similar exon structures. We have also identified the 21.5-kDa human MBP on immunoblots using antisera raised to a peptide encoded by the mouse exon 2 sequence. Southern blotting studies of human genomic DNA reveal a simple pattern consistent with a single human MBP gene. Thus, the three MBP mRNAs are likely to arise from alternative splicing of a primary human MBP transcript. Conservation of the 26 amino acid mouse exon 2 sequence in human MBP suggests an important role for this sequence in myelination.

Amino Acid Sequence↗
PubMed1986

Recombination within the myelin basic protein gene created the dysmyelinating shiverer mouse mutation.

Shiverer (shi) is an autosomal recessive mutation in the mouse characterized by an almost total lack of central nervous system myelin. While small amounts of other myelin components are present in the brain of the shi mouse, the four forms of myelin basic protein (MBP) are not detectable. Previous investigations by us and others indicate that the MBP gene has undergone a major rearrangement in the shi mutant. Herein, we report in detail the nature and extent of the rearrangement: a 20-kilobase region within the MBP gene is missing in the mutant. We map the 5' breakpoint of the deletion to the second intron and the 3' breakpoint to a site 2 kilobases beyond the last MBP exon. The junction of the upstream and downstream portions of the gene contains only one nucleotide not accounted for by the wild-type MBP gene sequence. The 3' side of the deletion occurs in the 3rd of 11 tandem repeats of a 31-base-pair sequence. This region is rich in alternating purine and pyrimidine stretches, sequences that have been associated with both Z-DNA structures and gene rearrangements. The recombination junction shares several features with the junctions characterized by Anderson et al. [Anderson, R., Kato, S. & Camerini-Otero, D. (1984) Proc. Natl. Acad. Sci. USA 81, 206-210] in mouse L cells and is consistent with their model for a partially homologous recombination event. The structure of the shi recombination junction suggests that the donor DNA molecules were aligned in a partially homologous region before staggered cutting and joining occurred.

Animals↗
PubMed1985

Alternative splicing accounts for the four forms of myelin basic protein.

We have isolated cDNA clones encoding the four different forms of mouse myelin basic protein (MBP) and have analyzed the structure of the MBP gene. The three larger forms of MBP differ from the smallest by the inclusion of either or both of two short amino acid sequences at positions 57 and 124 of the smallest protein. The mouse genome contains a single MBP gene comprised of seven exons. The two amino acid sequences present only in the larger MBPs are encoded by separate exons. Furthermore, all exons in the coding region begin or end in complete codons so that alternative splicing does not alter the reading frame. We conclude that the four forms of this myelin protein are encoded in separate mRNAs, each derived by a simple alternative splicing of the primary MBP gene transcript. Comparison of the amino acid sequence encoded by each exon with a recent model of the secondary structure of MBP suggests that each of the seven exons encodes one or two of the predicted structural motifs of the protein.

Amino Acid Sequence↗
PubMed1984

Correlation between growth inhibition and presence of 5'-methylthioadenosine in cells treated with interferon.

Treatment with interferon of several lines of human and murine cells resulted in a modest increase of the S- adenyosylmethionine level and in a greater increase of the S-adenosylhomocysteine level, with a consequent decrease in the S- adenyosylmethionine /S-adenosylhomocysteine ratio. These results confirmed a previous report (F. de Ferra and C. Baglioni , J. Biol. Chem., 258: 2118-2121, 1983) of such an effect of interferon on HeLa cells. The increase in S- adenyosylmethionine and S-adenosylhomocysteine was not observed in lymphoblastoid Raji cells, which are not growth inhibited by interferon. When Daudi cells and two other cell lines sensitive to growth inhibition by interferon were labeled with [35S]methionine, two novel labeled compounds were detected by high-pressure liquid chromatography analysis of cell extracts. These compounds were not present in untreated cells, and were either absent or present in reduced amount in extracts of cells resistant to the antiproliferative effect of interferon. One of the labeled compounds was tentatively identified with 5'-methylthioadenosine (MTA), a naturally occurring nucleoside, which was previously shown to inhibit murine lymphoid cell proliferation. Addition of MTA to cultures of different cell lines resulted in inhibition of cell growth. When the MTA concentration was measured in Daudi cells treated in this way, a relatively low concentration of MTA was detected, which was comparable to that found in interferon-treated cells. It seems possible that the presence of MTA in interferon-treated cells contributes to the inhibition of cell growth.

Adenosine↗
PubMed1983

Increase in S-adenosylhomocysteine concentration in interferon-treated HeLa cells and inhibition of methylation of vesicular stomatitis virus mRNA.

A fraction of the viral mRNA synthesized in interferon-treated HeLa cells infected with vesicular stomatitis virus (VSV) lacks the 7-methyl group in the 5'-terminal guanosine of the cap; this mRNA is not associated with polyribosomes and does not bind to ribosomes in an assay for initiation of protein synthesis (de Ferra, F., and Baglioni, C. (1981) Virology 112, 426-435). To establish whether this defect in methylation is due to changes in the level of the methyl donor S-adenosylmethionine (AdoMet) and of its competitive inhibitor S-adenosylhomocysteine (AdoHcy), we measured the concentration of these compounds in HeLa cells treated with interferon. An increase in both AdoMet and AdoHcy was detected 3 to 6 h after addition of interferon. The level of these compounds increased gradually and in proportion to the interferon concentration used. With 125 reference units/ml of beta interferon, for example, the AdoHcy concentration increased more than 3-fold and that of AdoMet about 1.5-fold with a consequent change in the AdoHcy/AdoMet ratio. An increased AdoHcy/AdoMet ratio was also found in HeLa cells treated with pure alpha 2 interferon produced in Escherichia coli by recombinant DNA techniques. When the methylation of VSV mRNA was measured in assays carried out with permeabilized virions at the AdoHcy and AdoMet concentrations found in interferon-treated cells, a preferential inhibition of the viral (guanine-7-)methyltransferase activity was observed. Such an inhibition may account for the synthesis of VSV mRNA lacking the 7-methyl group of guanosine in the cap.

Chromatography, High Pressure Liquid↗