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Biomedical subjects

H Murialdo

Publications and source records attributed to H Murialdo.

At least 37 records · Page 2Linked to original sources
PubMed1987

The overproduction of DNA terminase of coliphage lambda.

An artificial operon containing the genes coding for the two subunits of lambda DNA terminase, Nul and A, has been constructed. Derivatives of plasmid pBR322 served as the cloning vehicles. The transcription is driven by the pL promoter of phage lambda, and translation of the terminase genes was made efficient by the replacement of the wild-type ribosome-binding sites for those of lambda genes cII and/or D. The operon also carries the oL operator, and this enables regulation of its expression by a thermosensitive repressor. The synthesis of genes Nul and A products is extremely efficient upon derepression. Within 40 min after induction of the operon, the two subunits comprise about 20% of the total cellular protein mass. Crude extracts prepared from these overproducing strains are at least 100 times more active than extracts prepared from induced lambda lysogens in both promotion of lambda DNA packaging and cosmid cleaving. The ability to produce highly concentrated terminase would enormously facilitate the study of its structure and mechanism of action. These extracts are also extremely useful in techniques such as lambda DNA packaging, cosmid mapping and cosmid linearization to improve efficiency of integration into mouse eggs.

Bacteriophage lambda↗
PubMed1987

Synthesis of a trans-acting inhibitor of DNA maturation by prohead mutants of phage lambda.

Bacteriophage lambda with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA. In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development. We have constructed plasmids that contain the lambda-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos. The DNA terminase genes are under control of a thermosensitive cI repressor. These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors. However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids. Therefore, these plasmids can mature in the absence of proheads and other head gene products. The plasmids are also able to complement mutants of lambda deficient in terminase and DNA maturation. However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature. These experiments show that, in the absence of proheads, phage lambda produces a trans-acting inhibitor of maturation. The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C. A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor. Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.

Bacteriophage lambda↗
PubMed1986

A region of the immunoglobulin-mu heavy chain necessary for forming pentameric IgM.

In order to define the molecular requirements for IgM pentamer formation, we have isolated several mutant hybridomas which produce predominantly monomeric IgM. For one such mutant, 102, we synthesized a cDNA clone of its mu-mRNA, and found an in-frame 39-bp deletion, which thus encodes a mu-chain lacking amino acids 550-562, a region spanning the fourth constant domain and the tail of the mu-chain. To prove that this deletion is sufficient to block pentamer formation, we used site-directed mutagenesis to construct a mu-gene lacking these 39 bp, and have shown that the expression of this altered mu-gene results in the production of monomeric IgM.

Amino Acid Sequence↗
PubMed1986

Analysis of hybridoma mutants defective in synthesis of immunoglobulin M.

Hybridoma mutants defective in the expression of IgM have been analyzed by molecular and somatic cell hybridization techniques. The frequency of kappa light-chain mutants in the hybridoma PC7 was much higher than for other cell lines. In contrast to the mutations which we observed previously, the kappa mutants examined here resulted from complete or partial deletion of the kappa gene. Mutants defective in mu chain synthesis were of more diverse types including deletions, gross rearrangements, and more subtle changes. One mutant containing a cis-acting mutation resulting in reduced expression of the mu chain had an associated partial duplication of the mu gene, while others making low or undetectable levels of mu had no gross alterations in genetic structure. The usefulness of this approach to the study of gene structure and expression is discussed.

Animals↗
PubMed1985

Analysis of cosmids using linearization by phage lambda terminase.

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Animals↗
PubMed1985

Amplification of the gene for histidyl-tRNA synthetase in histidinol-resistant Chinese hamster ovary cells.

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Site-directed cleavage of immunoglobulin gene segments by lymphoid cell extracts.

To study the enzyme(s) involved in the site-specific recombination of immunoglobulin (Ig) gene segments, we designed an assay to detect V-J joining in vitro. The DNA from a hybrid phage (lambda VJCK) containing the VK41 gene segment separated by a 6-kilobase spacer region from the entire J-CK sequence was incubated with lymphoid cell extracts and packaged in vitro. Phages carrying genomic deletions were selected by screening for ethylenediaminetetraacetic acid resistance. Although no site-specific V-J fusion events were detected, the packaging efficiency of lambda VJCK DNA was 10(2)- to 10(3)-fold lower than that of lambda DNA. This suggested the presence in the cell extracts of an endonucleolytic activity with a specificity for the mouse DNA sequences. To detect the endonuclease cleavage products, plasmids containing VK or JK gene segments were used as a DNA substrate and the products of the in vitro reaction were visualized by autoradiography in Southern blots. Double-stranded cleavages were observed to occur near the 5' end of each one of the five JK gene segments and near the 3' end of a VK gene segment. A plasmid containing the mouse I-A beta gene was found to be resistant to cleavage, thus confirming the specificity of the endonucleolytic activity for sequences associated with the mouse Ig gene segments.

Animals↗
PubMed1985

Mouse immunoglobulin gene rearrangements. The sequence of a nonexpressed lambda 3-chain gene.

A functionally defective lambda 3-immunoglobulin chain gene has been cloned from plasmacytoma HOPC-1 (gamma 2b, lambda 1). The lambda 3 gene resulted from the juxtaposition of the germline V lambda 1 sequence with a J lambda 3 C lambda 3 gene segment. DNA sequencing of the rearranged V lambda 1 J lambda 3 exon showed the presence of a single base pair deletion at the site of V-J joining. The alteration in the reading frame caused by this deletion generated a stop codon at the 3' end of J lambda 3, thus rendering this gene nonfunctional for light chain production. In addition, a one-point mutation in the J lambda 3-C lambda 3 intron distinguishes the rearranged gene from the unrearranged counterpart. The implications that this rearrangement has in terms of the mechanism of somatic mutations and of selective proliferation of B cells mediated by antigen stimulation are discussed.

Animals↗
PubMed1984

Bacteriophage lambda preconnectors. Purification and structure.

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3 and E, and the two Escherichia coli genes groEL and groES . It has been shown previously that extracts prepared from cells infected with a lambda C-E- mutant accumulate a gpB polymer, which behaves as a biologically active intermediate in prohead assembly. This gpB activity has been called a preconnector , as it is probably a precursor to the head-tail connector. We now report the partial purification of biologically active preconnectors and the characterization of its structure. In the electron microscope, preconnectors appear as donut -like structures composed of several subunits displaying radial symmetry. Optical filtration of periodic arrays of preconnectors showed that the structure has 12-fold rotational symmetry. Side views of the preconnector reveal that it resembles an asymmetrical dumbell . This information has been used to construct a three-dimensional model of the preconnector . The implications of this structure for prohead shape and function, and for DNA packaging are discussed.

Bacteriophage lambda↗
PubMed1984

The maturation of coliphage lambda DNA in the absence of its packaging.

In vivo, lambda DNA cannot be cleaved at cos (matured) if proheads are not present; in vitro, however, cos cleavage readily takes place in the absence of proheads. In order to investigate this paradox, we have constructed plasmids that synthesize lambda terminase in vivo upon induction. The plasmids also contain cos at the normal position, about 190 bp upstream of lambda gene Nul. One of the plasmids, pFM3, produces levels of terminase comparable to those found after phage induction. If cells carrying pFM3 are thermoinduced, almost 100% of the intracellular plasmid DNA has a double-strand interruption at or near cos. Since the only lambda genes that pFM3 carries are Nul, A, W and B, this in vivo cleavage is occurring in the absence of proheads. Previous failure to observe lambda maturation with phages carrying prohead mutations may be due to exonucleolytic degradation of the unprotected DNA ends, a different DNA topology or compartmentalization, or terminase inhibition in the absence of prohead by the product of another lambda gene that maps to the right of gene B.

Bacteriophage lambda↗
PubMed1983

Nucleotide sequences of immunoglobulin mu heavy chain deletion mutants.

Mutants of an IgM producing hybridoma cell line were isolated which produce mu heavy chain fragments. Two such mutants were found to have internal deletions in the mu gene and the nucleotide sequence of the deletion endpoints was determined. No evidence was found for a role of the heavy chain switch region in the formation of these deletions. The implications of these mutants in defining the requirements of immunoglobulin gene expression are discussed.

Animals↗
PubMed1983

Early intermediates in bacteriophage lambda prohead assembly. II. Identification of biologically active intermediates.

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3, and E, as well as the E. coli genes groEL and groES. It has been previously shown that extracts prepared from cells infected with a lambda C-E- mutant accumulate biologically active gpB and gpNu3 (Murialdo, H., and Becker, A., J. Mol. Biol. 125, 57-74 (1978) ). To characterize the nature of these intermediates in prohead assembly, extracts prepared from these cells were fractionated by DEAE-cellulose chromatography as well as velocity sedimentation. Intermediates containing gpB were identified by SDS-polyacrylamide gel electrophoresis and by their ability to be assembled into biologically active proheads in vitro. The results indicate that the most abundant, biologically active intermediate (greater than 98% of the gpB activity) is a 25 S gpB-containing polymer. A second biologically active intermediate (about 1% of the total gpB activity) was identified as a gpB-gpgroEL complex.

Bacteriophage lambda↗
PubMed1983

Secretion of a lambda 2 immunoglobulin chain is prevented by a single amino acid substitution in its variable region.

We have studied two derivatives of the IgA (lambda 2) secreting myeloma cell line MOPC315:MOPC315.26, which produces and secretes a lambda 2 light chain, and MOPC315.37, which produces but does not secrete the lambda 2 chain. It has been reported that the only alteration in the MOPC315-37 lambda 2 chain is located in the variable region (Mosmann and Williamson, (1980) Cell 20, 283-292). In order to determine the nature of this alteration, we cloned the fragment of the chromosome containing the rearranged lambda 2 gene from both the nonsecreting variant MOPC315-37 and the normal lambda 2-secreting parent MOPC315-26 and determined their nucleotide sequence. We found that the nucleotide sequences coding for the leader peptide and for the constant region of the lambda 2 chain were identical in the secretor and nonsecretor. The sequences of the variable region differed at a single base pair corresponding to the first nucleotide in the codon for amino acid number 15. MOPC315-26 has a G in this position creating the codon GGT which codes for glycine, and MOPC315-37 has a C in this position creating the codon CGT which codes for arginine. Thus, we have demonstrated that a single amino acid substitution of a neutral amino acid, glycine, for a positively charged amino acid, arginine, results in the failure of a protein to be secreted.

Amino Acid Sequence↗
PubMed1982

Nucleotide sequence of a chromosomal rearranged lambda 2 immunoglobulin gene of mouse.

The rearranged lambda 2 gene of the mouse plasmacytoma cell line MOPC315 has been cloned and sequenced. A comparison of its sequence with the sequence of the unrearranged (germ-line) V, J and C gene segments shows that the sequences of the V gene segments differ at six positions. The sequence of the J and C gene segments remained unchanged. These results add support to the hypothesis that somatic mutations occur in immunoglobulin in genes and that these mutations do not involve the C gene segment. The degree of homology of the elements of the lambda 2 gene with those of the lambda 1 gene and C lambda 3 and C lambda 4 gene fragments suggest a pathway of evolution by gene duplication of the immunoglobulin lambda light chain locus. According to this scheme the original structure V0-J0C0 gave rise to a structure V0-J1C1-J11C11 by duplication of the J0C0 region. A second duplication encompassing the whole region resulted in the present structure: V1-J3C3-J1C1/V2-J2C2-J4C4.

Animals↗
PubMed1982

Orientation of cohesive end site cos determines the active orientation of chi sequence in stimulating recA . recBC-mediated recombination in phage lambda lytic infections.

Sequence chi, 5'G-C-T-G-G-T-G-G, locally enhances homologous recombination by recA and recBC proteins of Escherichia coli. Previous work showed that, in phage lambda, chi is more active in one orientation (leftward) than in the other (rightward). Inverting cos, the sequence for the mature DNA ends of lambda, reverses this orientation dependence: the rightward chi becomes fully active, and the leftward chi becomes relatively inactive. We surmise that chi action in phage lambda is coupled with DNA packaging or injection.

Bacterial Proteins↗
PubMed1982

Mutant immunoglobulin genes have repetitive DNA elements inserted into their intervening sequences.

The kappa light chain genes from two mutant hybridoma cell lines defective in kappa light chain synthesis were isolated and compared to the wild-type kappa light chain gene. In each case, the mutant kappa light chain genes were found to contain repetitive DNA elements in their intervening sequences that were not present in the intervening sequences of the wild-type kappa light chain gene. These elements were found to be related to the genes of intracisternal A particles. These results suggest that the decreased production of kappa light chain in the mutant cell lines is due to the presence of the intracisternal A particle-related genes.

Animals↗