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J M Calvo

Publications and source records attributed to J M Calvo.

At least 73 records · Page 4Linked to original sources
PubMed1985

Mutations that convert the four leucine codons of the Salmonella typhimurium leu leader to four threonine codons.

In Salmonella typhimurium, expression of the leucine operon is regulated by a transcription attenuation mechanism. According to a current model of attenuation, elevated expression of this operon requires that a ribosome stall at one of four adjacent codons for leucine on a leader RNA. We used oligonucleotide-directed mutagenesis to convert the four leucine codons of the S. typhimurium leu leader to four threonine codons. Analysis of the resulting mutant operon showed that almost all regulation by leucine had been abolished. The mutant operon was, instead, partially derepressed by a limitation for charged threonine tRNA. These results provide direct evidence for the function for the four leucine codons postulated by the attenuator model. An unexpected observation made during these studies was that the wild-type leu operon was partially derepressed by starvation for threonine.

Codon↗
PubMed1985

Unusual organization of the ilvIH promoter of Escherichia coli.

Analysis of plasmids containing ilvIH-galK fusions indicated that the Escherichia coli ilvIH promoter and sequences sufficient to cause leucine repression lie within 363 base pairs (bp) of ilvI. Experiments designed to locate the promoter and regulatory sequences more precisely gave the following results. The positions of the 5' endpoints of both unlabeled and pulse-labeled ilvIH mRNAs transcribed in vivo lie 30 bp upstream of ilvI. By contrast, the major in vitro RNA endpoints lie at positions further upstream. Several mutations which increase the expression of ilvIH lie 40 to 50 bp upstream of ilvI, within a putative promoter termed P1. Deletion of a 50-bp region immediately upstream of ilvI, which includes P1, resulted in the loss of all ilvIH promoter activity. Deletion of sequences more than 200 bp upstream of ilvI reduced ilvIH promoter activity by more than 80%. These results suggest that transcription of the ilvIH operon is initiated from promoter P1 but that sequences more than 200 bp upstream are required for optimal transcription of the operon.

Base Sequence↗
PubMed1984

The nucleotide sequence of a rat myosin light chain 2 gene.

A rat myosin light chain 2 gene was characterized by nucleotide sequence and S1 mapping analyses. It contains seven exons separated by six introns. The corresponding mRNA is predicted to be 654 nucleotides long (excluding polyA sequences), with 5'-nontranslated, coding, and 3'-nontranslated lengths of 56, 510, and 88 nucleotides, respectively. The predicted amino acid sequence is identical to that from rabbit except that the rat sequence lacks one of two Gly residues located at positions 12 and 13 in the rabbit sequence. From the nucleotide sequence, nascent rat myosin light chain 2 is predicted to have Met Ala preceding Pro at the N-terminal end.

Amino Acid Sequence↗
PubMed1984

Phasic activity of the basolateral amygdala, cingulate gyrus, and hippocampus during REM sleep in the cat.

We analyzed the electrical activity of the basolateral amygdala (BLA), anterior and posterior regions of the cingulate gyrus (A-CG and P-CG), the dorsal hippocampus (DH), the anterior ventral thalamic nucleus (AVTN), and the sensory motor cortex during the rapid eye movements and ponto-geniculo-occipital (PGO) activity of REM sleep in cats in chronic preparation. Polygraphic recordings and computational perievent averages using the phasic contractions of the lateral rectus muscle (LR) of the eyeball as the triggering signal of the analysis were performed. We observed biphasic potentials (200-300 ms) of variable amplitude, related to the phasic phenomena of REM sleep, in the BLA, A-CG, P-CG, DH, and AVTN. The latencies of the potentials of these regions were always greater than those of the geniculate PGO activities. We propose that the recorded limbic potentials resulted from propagation of PGO activity and that this phenomenon may reflect the limbic structure of the hallucinatory, vegetative, and emotional components of REM sleep.

Amygdala↗
PubMed1984

Promoter mutation causing catabolite repression of the Salmonella typhimurium leucine operon.

Two mutations that affect expression of the Salmonella typhimurium leu operon were investigated. leu operon DNA from these mutant strains was cloned, and nucleotide sequences of the leu control regions were determined. leu-500, which eliminates expression of all four leu genes simultaneously, is a point mutation in the -10 region of the leu promoter. leu-2012 is a point mutation within the -35 region of the leu promoter. leu-2012 suppressed leucine auxotrophy caused by leu-500 only when the medium contained a carbon source that does not cause catabolite repression. A cya mutation (adenylate cyclase deficiency) introduced into the leu-500 leu-2012 strain caused leu enzymes to be made only if cAMP was supplied exogenously. A leu-500 leu-2012 strain containing a crp mutation (cAMP receptor protein deficiency), on the other hand, could not make leu enzymes even in the presence of cAMP. In vitro transcription experiments demonstrated that the leu-2012 mutation created a new transcription initiation site. RNA polymerase utilized this site in vitro in the absence of added cAMP receptor protein and cAMP.

Base Sequence↗
PubMed1983

Transcription initiation sites of the leucine operons of Salmonella typhimurium and Escherichia coli.

Evidence for a transcription attenuation site downstream from the leu promoter was obtained by transcription experiments in vitro. Most transcription initiated in vitro from leuP is terminated prematurely, resulting in the synthesis of a 160 nucleotide leader RNA. We define here the point at which transcription is initiated in vitro and in vivo and demonstrate that the site of premature termination is between the promoter and the first structural gene (leuA). Additional nucleotide sequences are presented that extend the known sequence 200 base-pairs upstream and 300 base-pairs downstream from leuP. The location of the promoter-proximal end of cistron leuA was deduced by comparing nucleotide sequence data with the sequence of the ten amino acids at the N-terminus of alpha-isopropylmalate synthase. To facilitate the isolation of quantities of material for sequencing experiments, the enzyme was isolated from a plasmid-containing strain, CV605, grown under conditions of leucine limitation. Under such conditions, about 20% of the total soluble protein of strain CV605 is alpha-isopropylmalate synthase and another 20% is beta-isopropylmalate dehydrogenase (leuB product).

Amino Acid Sequence↗
PubMed1983

Molecular structure of ilvIH and its evolutionary relationship to ilvG in Escherichia coli K12.

ilvIH of Escherichia coli K12 codes for a valine-sensitive acetohydroxy acid synthase (AHASIII). The DNA sequence of ilvIH was determined. Open reading frames and appropriate translation signals exist for two polypeptides, one containing 565 amino acids (ilvI polypeptide) and the other 160 amino acids (ilvH polypeptide). A graphic matrix analysis shows three clearcut regions of homology between ilvI and ilvG (codes for AHASII). Within these three regions of homology, 50-60% of the amino acid sequences of AHASII and AHASIII are conserved. Inspection of the region between ilvG and ilvE (the K region) revealed that it can potentially code for an 86 amino acid polypeptide. A computer analysis shows small but significant homology between the predicted amino acid sequences of the N-terminal half of the ilvH polypeptide and the putative region K polypeptide. We conclude that ilvIH and ilvG-region K evolved from a common ancestor.

Acetolactate Synthase↗
PubMed1983

Transcription attenuation is the major mechanism by which the leu operon of Salmonella typhimurium is controlled.

Three mutations, each causing constitutive expression of the Salmonella typhimurium leu operon, were cloned into phage vector lambda gt4 on EcoRI DNA fragments carrying all of that operon except for part of the promoter-distal last gene. Sequence analysis of DNA from these phage demonstrated that each contains a single base change in the leu attenuator. Transcription of mutant DNA in vitro resulted in transcription beyond the usual site of termination. The level of beta-IPM dehydrogenase, the leuB enzyme, was elevated 40-fold in a strain carrying one of these mutations, and starvation of this strain for leucine had little effect on the amount of activity expressed. Using a strain with a wild-type promoter-leader region of the leu operon, the rates of synthesis and degradation of leu leader RNA and readthrough RNA (leu mRNA) were measured by DNA-RNA hybridizations with specific DNA probes. The rate of synthesis of the leu leader was about the same in cells grown with excess or with limiting leucine. On the other hand, the rate of synthesis of leu mRNA was 12-fold higher for cells grown in limiting leucine as opposed to excess leucine. The rate of degradation of these RNA species was the same under both conditions of growth. Thus, the variation in expression of the leu operon observed for cells grown in minimal medium is, for the most part, not caused by control over the frequency of initiation or by the differential stability of these RNA species. Rather, the variation is a direct result of the frequency of transcription termination at an attenuator site. These results taken together suggest that transcription attenuation is the major mechanism by which leucine regulates expression of the leu operon of S. typhimurium for cells growing in a minimal medium.

Bacteriophage lambda↗
PubMed1983

IlvHI locus of Salmonella typhimurium.

In Escherichia coli K-12, the ilvHI locus codes for one of two acetohydroxy acid synthase isoenzymes. A region of the Salmonella typhimurium genome adjacent to the leucine operon was cloned on plasmid pBR322, yielding plasmids pCV47 and pCV49 (a shortened version of pCV47). This region contains DNA homologous to the E. coli ilvHI locus, as judged by hybridization experiments. Plasmid pCV47 did not confer isoleucine-valine prototrophy upon either E. coli or S. typhimurium strains lacking acetohydroxy acid synthase activity, suggesting that S. typhimurium lacks a functional ilvHI locus. However, isoleucine-valine prototrophs were readily isolated from such strains after mutagenesis with nitrosoguanidine. In one case we found that the Ilv+ phenotype resulted from an alteration in bacterial DNA on the plasmid (new plasmid designated pCV50). Furthermore, a new acetohydroxy acid synthase activity was observed in Ilv+ revertants; this enzyme was similar to E. coli acetohydroxy acid synthase III in its lack of activity at low pH. This new activity was correlated with the appearance in minicells of a new polypeptide having an approximate molecular weight of 61,000. Strains carrying either pCV49 or pCV50 produced a substantial amount of ilvHI-specific mRNA. These results, together with results from other laboratories, suggest that S. typhimurium has functional ilvB and ilvG genes and a cryptic ilvHI locus. E. coli K-12, on the other hand, has functional ilvB and ilvHI genes and a cryptic ilvG locus.

Acetolactate Synthase↗
PubMed1982

Amplification and modification of dihydrofolate reductase in Escherichia coli. Nucleotide sequence of fol genes from mutationally altered plasmids.

Recombinant plasmids carrying the structural gene for Escherichia coli dihydrofolate reductase (fol) were mutagenized in vitro and in vivo and were used to transform a suitable recipient strain. Twenty-three transformants were isolated that were able to grow in the presence of high levels of the folate analog trimethoprim, and, in each strain, the resistance determinant was shown to be carried on the plasmid. Three of the strains produced dihydrofolate reductase with an increased Ki value for trimethoprim. DNA sequence analysis showed that the plasmids in these strains had mutations in fol which altered a conserved region of the polypeptide that forms part of the dihydrofolate-binding site. Two other strains had approximately 3-fold elevated dihydrofolate reductase levels, apparently resulting from plasmid copy number mutations. The remaining 18 strains had dihydrofolate reductase levels that were 10-30 times higher than those of the starting strain. Surprisingly, three of these strains had no discernible changes either in plasmid copy number or in the nucleotide sequence of the plasmid fol gene. Sequence analysis of the plasmids in 12 more of the strains revealed mutations in the promoter region adjacent to the fol gene. Most of these mutations occurred in the conserved sequences known as the Pribnow box and the -35 region and increased the homology of these sequences with the consensus E. coli promoter sequence. Strains carrying these plasmids produced a significant fraction of their total cell protein as wild type dihydrofolate reductase and should therefore be useful as sources of the purified enzyme.

Autoradiography↗
PubMed1982

Amygdaloid kindling during rapid eye movement (REM) sleep in cats.

Development of amygdaloid kindling was analyzed during REM sleep and during wakefulness. Daily evolution of electrographic and behavioral changes was significantly delayed in REM kindled rats. The number of kindling trials required to reach the first generalized convulsive seizure was also significantly increased in comparison with awake kindled animals. Changes in sleep organization were measured under REM kindling conditions. A significant increase in total sleep time and in the percentage of light slow-wave sleep was found during the kindling process. No significant sleep changes were observed in REM-established kindling. REM inhibitory influence over epileptogenesis is discussed.

Amygdala↗
PubMed1982

Nucleotide sequence of dihydrofolate reductase genes from trimethoprim-resistant mutants of Escherichia coli. Evidence that dihydrofolate reductase interacts with another essential gene product.

We report the construction of recombinant plasmids containing the dihydrofolate reductase structural gene (fol) from several trimethoprim-resistant mutants of Escherichia coli. Strains carrying some of these plasmids produced approximately 6% of their soluble cell protein as dihydrofolate reductase and are therefore excellent sources of the purified enzyme for inhibitor binding or mechanistic studies. The nucleotide sequence of the fol region from each of the plasmids was determined. A plasmid derived from a Ki mutant which produced a dihydrofolate reductase with lowered affinity for trimethoprim contained a mutation in the structural gene that altered the sequence of the polypeptide in a conserved region which is adjacent to the dihydrofolate binding site. Two other independently-isolated mutants which overproduced dihydrofolate reductase had a mutation in the -35 region of the fol promoter. One of them, strain RS35, was also temperature-sensitive for growth in minimal medium. This phenotype was shown to be the result of an additional mutation in a locus unlinked to fol by P1 transduction. The fol regions from two temperature-independent revertants of strain RS35 were sequenced. One of these had a mutation within the dihydrofolate reductase structural gene which altered some properties of the enzyme. This confirmed some previous enzymological data which suggested that some revertants of strain RS35 had mutations in fol (Sheldon 1977). These results suggest that dihydrofolate reductase interacts physically with some other essential gene product in E. coli.

Base Sequence↗
PubMed1982

Kindling in the spinal cord: differential effects on mono- and polysynaptic reflexes and its modifications by atropine and naloxone.

We have used the kindling paradigm at the spinal cord level. In spinal, unanaesthetized, paralysed (gallamine 20 mg/kg) cats, the cutaneous afferent (sural) or the muscle afferent (gastrocnemius, tibial and peroneal) nerves were electrically stimulated at 20 min intervals with a 3 sec train (100 Hz, 2 msec pulse duration, 200-400 microA). Kindling was assessed by averaging 16 mono- or polysynaptic ventral root reflex responses produced by constant intensity single shocks. Atropine sulphate (0.5 mg/kg) and naloxone (0.4 mg/kg) effects were measured in the kindled preparation. Kindling induced a progressive increment of the amplitude, ipsilateral propagation and after-discharge frequency. This was greater in polysynaptic responses. When testing monosynaptic responses during the kindling of cutaneous afferents, a cumulative inhibitory effect was observed. In both types of kindled response, atropine had a transient inhibitory effect. Naloxone noticeably augmented the kindled polysynaptic reflexes.

Animals↗
PubMed1981

Physical characterization of the ilvHI operon of Escherichia coli K-12.

The ilvHI and leu genes of Escherichia coli K-12 are contained on a single 10.9-kilobase EcoRI fragment of deoxyribonucleic acid derived from the leu transducing phage lambda G4. Since the expression of all of these genes is controlled by leucine, we investigated whether they are part of single operon or whether they constitute separate but adjacent operons controlled from a common site. Both cloning and hybridization studies indicated that ilvHI and leu are distinct operons. They are transcribed in opposite directions and are separated by approximately 1,500 base pairs of deoxyribonucleic acid. Hybridization experiments showed that the expression of ilvHI is regulated chiefly at the level of transcription. The size of the ilvHI messenger ribonucleic acid is estimated to be 2,550 bases.

Acetolactate Synthase↗
PubMed1980

Nucleotide sequence of the E coli gene coding for dihydrofolate reductase.

Plasmid pLC1437a contains DNA from Escherichia coli K12 including fol, the structural gene for dihydrofolate reductase. The fol gene was mapped on this plasmid relative to several restriction endonuclease cleavage sites. fol was also cloned from strain RSO and the nucleotide sequence for the entire fol gene and its flanking regions from this strain was determined. The amino acid sequence predicted from the nucleotide sequence differs in only a few respects from the reported amino acid sequence of dihydrofolate reductase from E. coli B. The major RNA transcripts initiated at the fol promotor in vivo are approximately 550 and 590 nucleotides long. In addition to these, several longer transcripts (up to 1400 nucleotides) are present in lesser amounts. A new procedure is described for 3' end labeling of DNA fragments having blunt ends using E. coli exonuclease III and avian myeloblastoma virus reverse transcriptase.

Base Sequence↗
PubMed1980

Cloning of an EcoRI-generated fragment of the leucine operon of Salmonella typhimurium.

Recombinant plasmids carrying part of the leucine operon of Salmonella typhimurium were isolated following transformation of an Escherichia coli leucine auxotroph to prototrophy with a ligated mixture of EcoRI-treated Salmonella DNA and plasmid pSC101 DNA. Plasmids pCV11 and pCV13, containing a 3.4-10(6) dalton DNA fragment ligated to the vector, had the leu operon oriented in opposite directions. The orientation of the leu operon relative to plasmid genes was determined. The 3.4-10(6) dalton fragment was ligated in to the EcoRI site of plasmid pMB9 yielding plasmids pCV12 (orientation as in pCV11) and pCV14 (orientation as in pCV13). The results of enzyme assays and complementation tests indicated that these plasmids carry functional leuA, leuB, and leuC genes but not a functional leuD gene. Furthermore, the following results indicated that they have a functional leu control region and promoter. Expression of plasmid leu genes was markedly enhanced under conditions of leucine limitation whereas introduction of a leu promoter mutation into the operon oriented in either direction with respect to plasmid genes had a strong negative effect upon leu operon expression. Transcriptional readthrough from plasmid promoters, if it occurs at all, must be small in comparison with transcription initiated at the leu promoter. RNA was isolated from leucine auxotrophs grown under conditions of repression and derepression and from prototrophic strains derepressed for the leucine operon as a result of mutations in leuO, leuS, and flrB. The rate of synthesis of leu mRNA, measured by hybridization to plasmid pCV12 DNA, was proportional in each case to leu enzyme levels.

Cloning, Molecular↗
PubMed1979

Regulation of dihydrofolate reductase synthesis in Escherichia coli.

Two clones from the Clarke-Carbon Escherichia coli colony bank were resistant to inhibition by trimethoprim, a potent inhibitor of dihydrofolate reductase. Both clones had elevated levels of dihydrofolate reductase. Furthermore, trimethoprim resistance and elevated enzyme levels were associated with ColE1 plasmids that carried DNA from the trkC ksgA pdxA region of the E. coli chromosome. Plasmid pLC1437a was shown by two criteria to carry the structural gene for dihydrofolate reductase: 1) A partial diploid containing plasmid pLC1437a produced a kinetically-recognizable dihydrofolate reductase that was not present in the parent haploid strain. 2) Plasmid pLC1437a coded for dihydrofolate reductase in vitro. A 1,000 base pair fragment of plasmid pLC1437a containing fol was used as a probe to measure fol mRNA in a mutant strain isolated by Sheldon and Brenner (Molec. gen. Genet. 147, 91-97, 1976). The mutation in this strain, which results in constitutively-high levels of dihydrofolate reductase and in the inability of the strain to grow at 42 degrees C, is cis dominant (Sheldon and Brenner, 1976). The results of kinetic hybridization and pulse-labeling experiments indicated that the regulatory mutant produced elevated levels of dihydrofolate reductase in response to an increased rate of synthesis of fol mRNA.

DNA Restriction Enzymes↗