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Biomedical subjects

J E Brooks

Publications and source records attributed to J E Brooks.

At least 19 recordsLinked to original sources
PubMed1992

Highly repeated DNA sequences in birds: the structure and evolution of an abundant, tandemly repeated 190-bp DNA fragment in parrots.

Up to 6.8% of the parrot (Psittaciformes) genome consists of a tandemly repeated, 190-bp sequence (P1) located in the centromere of many if not all chromosomes. Monomer repeats from 10 different psittacine species representing four subfamilies were isolated and cloned. The intraspecific sequence variation ranged from 1.5 to 7%. The interspecific sequence variation ranged from less than 3% between two species of cockatoos to approximately 45% between cockatoos and other parrots. The monomer sequences of all 10 parrot species contained several conserved (> 90%) sequence elements at identical locations within the repeat. A comparison with tandemly repeated DNA sequences in other avian species showed that several of these conserved elements were also present at similar locations within the 184-bp repeat of the Chilean flamingo (Phoenicopterus chilensis), suggesting a great antiquity of the repeat. One of the elements was also found in the tandemly repeated sequences of the crane (Gruidae) and falcon (Falconidae) families. The data were used for the construction of a partial most parsimonious relationship that supports a regional subdivision of the Psittaciformes.

Animals↗
PubMed1992

Regulation of the BamHI restriction-modification system by a small intergenic open reading frame, bamHIC, in both Escherichia coli and Bacillus subtilis.

BamHI, from Bacillus amyloliquefaciens H, is a type II restriction-modification system recognizing and cleaving the sequence G--GATCC. The BamHI restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene. The small open reading frame has been designated bamHIC (for BamHI controlling element). It acts as both a positive activator of endonuclease expression and a negative repressor of methylase expression of BamHI clones in Escherichia coli. Methylase activity increased 15-fold and endonuclease activity decreased 100-fold when bamHIC was inactivated. The normal levels of activity for both methylase and endonuclease were restored by supplying bamHIC in trans. The BamHI restriction-modification system was transferred into Bacillus subtilis, where bamHIC also regulated endonuclease expression when present on multicopy plasmid vectors or integrated into the chromosome. In B. subtilis, disruption of bamHIC caused at least a 1,000-fold decrease in endonuclease activity; activity was partially restored by supplying bamHIC in trans.

Bacillus subtilis↗
PubMed1991

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.

Amino Acid Sequence↗
PubMed1989

Cloning the BamHI restriction modification system.

BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence of the DdeI restriction-modification system and characterization of the methylase protein.

The DdeI restriction-modification system was previously cloned and has been maintained in E. coli on two separate and compatible plasmids (1). The nucleotide sequence of the endonuclease and methylase genes has now been determined; it predicts proteins of 240 amino acids, Mr = 27,808, and 415 amino acids, Mr = 47,081, respectively. Inspection of the DNA sequence shows that the 3' end of the methylase gene had been deleted during cloning. The clone containing the complete methylase gene was made and compared to that containing the truncated gene; only clones containing the truncated form support the endonuclease gene in E. coli. Bal-31 deletion studies show that methylase expression in the Dde clones is also dependent upon orientation of the gene with respect to pBR322. The truncated and complete forms of the methylase protein were purified and compared; the truncated form appears to be more stable and active in vitro. Finally, comparison of the deduced amino acid sequence of M. DdeI with that of other known cytosine methylases shows significant regions of homology.

Amino Acid Sequence↗
PubMed1987

Properties and uses of restriction endonucleases.

It is clear that we have still not exhausted all the restriction endonuclease specificities to be found in nature. Recently discovered BsmI is the first endonuclease recognizing a nonpalindromic sequence that cleaves within the site. Certainly other endonucleases belonging to this class will soon be discovered. More endonucleases are now being sought that recognize longer recognition sequences, because large fragments can now be readily separated by pulse-field electrophoresis. New sources of endonucleases are also being found; for example, a group of viruses that grow on Chlorella algae produce type II-like site-specific endonucleases. As the number and variety of known restriction endonucleases increase, the number and variety of applications keep pace. There is still no end in sight.

Base Sequence↗
PubMed1986

Cloning the DdeI restriction-modification system using a two-step method.

DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.

Bacteriophage lambda↗
PubMed1986

Prolactin and stress: some endocrine correlates of pre-operative anxiety.

An attempt was made to correlate the serum concentration of PRL, GH and cortisol with anxiety experienced before elective surgery. A significant direct correlation was observed between cortisol and anxiety (P = 0.028), and between GH and anxiety (P = 0.001) although the latter correlation may have reflected the confounding effects of sex and age on GH levels. No correlation was observed between anxiety and PRL and we conclude that psychosocial stress cannot be invoked as an occasional cause of hyperprolactinaemia.

Aged↗
PubMed1985

Nucleotide sequence of the PaeR7 restriction/modification system and partial characterization of its protein products.

Bal31 deletion experiments on clones of the PaeR7 restriction-modification system from Pseudomonas aeruginosa demonstrate that it is arranged as an operon, with the methylase gene preceding the endonuclease gene. The DNA sequence of this operon agrees with in vitro transcription-translation assays which predict proteins of 532 amino acids, Mr = 59,260 daltons, and 246 amino acids, Mr = 27,280 daltons, coincident with the methylase and endonuclease genes, respectively. These predicted values coincide with the measured molecular weights of the purified, denatured PaeR7 endonuclease and methylase proteins. The first twenty amino acids from the amino-terminus of the purified endonuclease exactly match those predicted from the DNA sequence. Finally, potential regulatory mechanisms for the expression of phage restriction are described based on the properties of several PaeR7 subclones.

Amino Acid Sequence↗
PubMed1978

Laboratory evaluation of pyriminyl used as a rodenticide against the lesser bandicoot rat, Bandicota bengalensis.

The properties of pyriminyl (N-3-pyridylmethyl-N'-p-nitrophenyl urea) as a rodenticide against the lesser bandicoot rat (Bandicota bengalensis) in Rangoon, Burma, were investigated in the laboratory. The acute LD 50 and LD 95 dose of orally administered pyriminyl for B. bengalensis were found to be 6.7 mg/kg and 23.0 mg/kg of body weight respectively. When caged bandicoots were given a choice between plain and poisoned baits, the optimum rodenticidal concentration in the bait was found to be 0.25-0.5%. Symptoms of pyriminyl poisoning appear from 1 to 4 h after feeding starts, giving individual animals time to consume from 2 to over 30 LD 50 doses of 0.5% pyriminyl before feeding stops. Deaths occurred from 4 to 96 h after either oral dosing or free-choice feeding. There appeared to be no significant aversion to the poison at 0.25% or 0.5% concentration in foods. The potential hazards and use of pyriminyl as a field bait against populations of B. bengalensis are discussed.

Administration, Oral↗
PubMed1975

Excitation-contraction uncoupling. The effect of hyperosomolar glycerol solution and antrolene sodium on mammalian muscle in vitro.

In the rat hemidiaphragm, in vitro dantrolene sodium irreversibly reduced twitch tension up to 20% of the original value in 20 minutes of exposure. The phenomenon was dose related. Dantrolene sodium did not affect (1) resting membrane potential (IMP); (2) miniature endplate potential frequency of amplitude; or (3) endplate potential amplitude decrement of repetitively stimulated partially curarized muscle. A combination of hyperosmolar solutions of glycerol and dantrolene sodium abolished contraction of mammalian muscle with no demonstrable effect on neuromuscular transmission or muscle fiber RMP. With this technique, prolonged recording from repetitively activated muscle fibers is possible.

Action Potentials↗
PubMed1975

The mechanism of neuromuscular fatigue. A study of mammalian muscle using excitation-contraction uncoupling.

Neuromuscular transmission failure was measured during rapid repetitive stimulation of the rat hemidiaphragm in vitro after excitation-contraction uncoupling by hyperosmolar glycerol, washing, and dantrolene sodium. Observations of resting membrane, miniature endplate, action, and endplate potentials (epps) showed that neuromuscular transmission failure occurred during stimulation because of a decrement of the epp. This decrement was due both to a reduced number of quanta released and to a variable reduction of quantal size. The time course of these events was studied, and limited observations were made on recovery. Observations of the difference between the epp decrement of cut muscle and of partially curarized muscle suggest a prejunctional action of curare.

Action Potentials↗