Search PubMed⌕ Search

Biomedical subjects

J McLauchlan

Publications and source records attributed to J McLauchlan.

At least 37 records · Page 2Linked to original sources
PubMed1991

Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli.

The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in Escherichia coli. The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3.5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5.3. Atomic emission spectroscopy indicated that the iron content of the E. coli-expressed R2 was 0.2 to 0.5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The E. coli-expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the E. coli-expressed R2 with the E. coli-expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR.

Amino Acids↗
PubMed1990

Insertion of DNA sequences at a unique restriction enzyme site engineered for vector purposes into the genome of herpes simplex virus type 1.

We describe the construction of a novel herpes simplex virus (HSV) vector containing a unique XbaI restriction enzyme cloning site in an intergenic position in the short unique genome region. Sequences can be inserted at this site with high efficiency by ligation with XbaI-digested vector DNA. A series of plasmids has been designed for use with this vector. These allow protein coding sequences to be placed under the control of various transcriptional regulation signals and then isolated as XbaI fragments ready for insertion into the vector. The XbaI fragments also contain the beta-galactosidase gene thereby facilitating selection of recombinant virus by screening for blue plaques. A variant of the vector has been made based on the temperature-sensitive (ts) mutant tsK, which expresses only immediate early (IE) genes at non-permissive temperatures. Chloramphenicol acetyltransferase was used as a reporter gene to assess the fidelity of expression of sequences cloned into this position. Under these circumstances IE and early HSV promoters were shown to behave as expected in both wild-type and ts vectors.

Animals↗
PubMed1989

Herpes simplex virus induces a processing factor that stimulates poly(A) site usage.

Extracts from herpes simplex virus-infected cells and from mock-infected cells have been compared for their ability to process at RNA poly(A) sites in vitro. Nuclear extracts from infected cells contain an activity that increases processing efficiency specifically at a late herpes simplex virus poly(A) site. By contrast, a second virus poly(A) site is processed with equal efficiency by nuclear extracts from infected and mock-infected cells. Using precursor RNAs containing these two virus poly(A) sites in tandem, which allows ready detection of the processing factor, we show that this specific activity is heat labile. Analysis of RNAs produced by virus recombinants that contain the poly(A) site sequences in tandem also indicates that increased processing at the late virus poly(A) site occurs in vivo.

Base Sequence↗
PubMed1988

Components required for in vitro cleavage and polyadenylation of eukaryotic mRNA.

We have studied in vitro cleavage/polyadenylation of precursor RNA containing herpes simplex virus type 2 poly A site sequences and have analyzed four RNA/protein complexes which form during in vitro reactions. Two complexes, A and B, form extremely rapidly and are then progressively replaced by a third complex, C which is produced following cleavage and polyadenylation of precursor RNA. Substitution of ATP with cordycepin triphosphate prevents polyadenylation and the formation of complex C however a fourth complex, D results which contains cleaved RNA. A precursor RNA lacking GU-rich downstream sequences required for efficient cleavage/polyadenylation fails to form complex B and produces a markedly reduced amount of complex A. As these GU-rich sequences are required for efficient cleavage, this establishes a relationship between complex B formation and cleavage/polyadenylation of precursor RNA in vitro. The components required for in vitro RNA processing have been separated by fractionation of the nuclear extract on Q-Sepharose and Biorex 70 columns. A Q-Sepharose fraction forms complex B but does not process RNA. Addition of a Biorex 70 fraction restores cleavage activity at the poly A site but this fraction does not appear to contribute to complex formation. Moreover, in the absence of polyethylene glycol, precursor RNA is not cleaved and polyadenylated, however, complexes A and B readily form. Thus, while complex B is necessary for in vitro cleavage and polyadenylation, it may not contain all the components required for this processing.

Base Sequence↗
PubMed1986

Structural features of ribonucleotide reductase.

Herpes simplex virus type 1 (HSV-1) encodes a ribonucleotide reductase which comprises two polypeptides with sizes of 136,000 (RR1) and 38,000 mol. wt. (RR2). We have determined the entire DNA sequence specifying HSV-1 RR1 and have identified two adjacent open reading frames in varicella-zoster virus (VZV) which have homology to HSV RR1 and RR2; the predicted sizes for the VZV RR1 and RR2 polypeptides are 87,000 and 35,000 mol. wt. respectively. Amino acid comparisons with RR1 and RR2 polypeptides from other organisms indicate that HSV-1 RR1 contains a unique N-terminal domain which is absent from other RR1 polypeptides apart from HSV-2 RR1. These N-terminal amino acid sequences are poorly conserved between HSV-1 and HSV-2 in contrast to the remainder of the protein which shows greater than 90% homology. Polypeptide structural predictions suggest that the HSV-1 N-terminal domain may be separated into two regions, namely, a beta-sheet structure followed by a nonstructured area. Across the remainder of RR1 and RR2, comparisons also reveal blocks of amino acids conserved between the different ribonucleotide reductases, and these may be important for enzyme activity. From predictions on the structure of these conserved blocks, we have proposed that the location of a substrate binding site within RR1 is centered on three conserved glycine residues in a region which is predicted to adopt a beta-sheet/turn/alpha-helical structure; this approximates to the structure for ADP nucleotide binding folds. Finally, we propose that the promoters for the HSV and Epstein-Barr virus (EBV) RR2 transcripts have evolved by separate evolutionary routes.

Amino Acid Sequence↗
PubMed1985

A modular system for the assay of transcription regulatory signals: the sequence TAATGARAT is required for herpes simplex virus immediate early gene activation.

A modular system for assaying the activity of transcriptional regulatory signals based on herpes simplex virus (HSV) promoter and terminator sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene has been used to study activation of HSV immediate early (IE) gene expression. Insertion of the SV40 72 base pair (bp) repeat increased mRNA levels by 15-fold thus demonstrating the ability of the HSV IE promoter to respond to a heterologous enhancer. A fragment containing part of the intergenic region located between HSV-2 immediate early (IE) genes-3 and -4/-5 increased mRNA levels by 5-fold in response to transactivation by an HSV virion structural polypeptide. The HSV activator fragment increased mRNA levels by 2-fold in the absence of transactivation indicating that cellular proteins are involved in IE gene expression. From HSV-1/HSV-2 DNA sequence comparisons we previously proposed that a DNA sequence, consensus TAATGARAT, present upstream of all HSV-1 and HSV-2 IE genes was required for the co-ordinate induction of IE genes. We show here that a synthetic oligonucleotide containing TAATGARAT conferred the ability to stimulate CAT activity only on transactivation: two copies of TAATGARAT stimulated expression by 2-fold while six copies gave an 8-fold increase. This activation, which was not dependent on orientation of the TAATGARAT sequence, directly demonstrates that TAATGARAT is a component of the IE gene activation sequence.

Acetyltransferases↗
PubMed1985

The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3' termini.

Our previous DNA sequence comparisons of 3' terminal portions from equivalent herpes simplex virus type 1 (HSV-1) and HSV-2 genes identified a conserved sequence (consensus YGTGTTYY; Y = pyrimidine) located approximately 30bp downstream from the AATAAA signal. We report here that this signal is located downstream from 67% of the mammalian mRNA 3' termini examined. Using constructions with the bacterial chloramphenicol acetyl transferase (CAT) gene linked to an HSV 'terminator' fragment, we show that deletions in the 'terminator' reduce CAT activities and the levels of CAT mRNA 3' termini. Specifically: (1) deletions of downstream sequences which extend up to the consensus YGTGTTYY signal reduce CAT levels to values 35% of those obtained with undeleted plasmids, (2) a deletion of a further 14bp, which removes the YGTGTTYY consensus but not the poly A site, reduces CAT activities to 1%-4%. The levels of CAT mRNA 3' termini reflect the reductions in CAT activities however, levels of mRNA 5' termini are unaffected by these deletions. The RNA produced in the absence of the YGTGTTYY signal is present in the cytoplasm although no CAT activity is detectable.

Acetyltransferases↗
PubMed1984

The recovery of quadriceps function in patients treated by cast bracing.

The recovery of quadriceps function in patients treated by cast bracing has been assessed in fifty-four patients. The isometric strength and dynamic endurance of the quadriceps muscle were measured at an average of 2 years 8 months after the fracture. The literature is reviewed and a simple method of measurement is described. The results indicate that the recovery of quadriceps function after cast bracing in an unselected group of patients compares favourably with that after open and closed nailing techniques.

Adolescent↗
PubMed1984

The stracathro approach to the hip.

A new approach to the hip joint is described in which the integrity of the gluteus medius is preserved. It provides good exposure of the hip while causing the least disturbance to the abductor mechanism.

Bursitis↗
PubMed1983

DNA sequence homology between two co-linear loci on the HSV genome which have different transforming abilities.

A transcription unit at the herpes simplex virus (HSV) type 2 transforming region, mtr-2 (map coordinates 0.580-0.625), comprises two early, unspliced mRNAs of 4.5 kb and 1.2 kb which are 3' co-terminal; a region including that specifying the 1.2-kb mRNA has been sequenced. The putative translated portions of these two mRNAs do not overlap and this feature, together with the arrangement of the mRNAs, is similar to the apparently equivalent co-linear HSV-1 locus which however does not transform. A putative stem and loops structure containing a TATA box is located upstream from the 5' terminus of the 1.2-kb mRNA within the translated portion of the 4.5-kb mRNA. Evidence for the generation of this structure by intra-strand reassociation under our hybridisation conditions has been obtained and possibilities are that it may function as a transcription-activated promoter or as an RNA polymerase pause site. A comparison of the equivalent HSV-2 and HSV-1 regions reveals a conserved sequence downstream from the 3' co-terminus which is present at a similar location in many eukaryotic genes (consensus sequence YGTGTTYY). The overall sequence conservation at this transcription unit is high except for regions located at: (1) the untranslated leaders of the 1.2-kb mRNAs; (2) the N termini of the polypeptides specified by the HSV-2/HSV-1 1.2-kb mRNAs; (3) the intergenic region beyond the 3' co termini. Regions (2) and (3) are located within a transforming fragment of HSV-2. The possible significance of these data for HSV-mediated cell transformation is discussed.

Amino Acid Sequence↗
PubMed1983

Organization of the herpes simplex virus type 1 transcription unit encoding two early proteins with molecular weights of 140000 and 40000.

The 5' ends of two early herpes simplex virus type 1 mRNAs have been identified by nuclease S1 and exonuclease VII analysis using cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region between map coordinates 0.56 and 0.60, are unspliced and share a 3' terminus. Genomic DNA at the 5' ends has been sequenced and the 5' termini have been located on the virus DNA sequence. The DNA sequence has revealed signals involved in the initiation of transcription of both mRNAs, and the 5' end of the 1.2 mRNA is encoded within the internal sequences of the 5.0 kb mRNA. The probable translational initiation codons for the polypeptides specified by these mRNAs have been identified, and the results indicate that the coding regions of the two mRNAs do not overlap.

Base Sequence↗
PubMed1982

Sterile microenvironment in prevention of wound infection.

A prospective controlled trial was carried out to assess the effect of using a wound isolator on reducing postoperative infection. A total of 291 patients undergoing hip pinning for fractures of the neck of femur entered the trial and were allocated at random to have their wound contained in a wound isolator (study group) or dressed with a standard gamma-irradiated adhesive dressing (control group). The bacteriological flora of the patient was monitored before, during, and after operation and that of the ward before and after. No significant difference was found in the flora of the wards in which the patients were nursed. On several occasions the source of the infective organism was traced to the ward but never to the theatre. The isolator prevented direct contamination and airborne cross-infection of the wound and appreciably reduced the rate of infection.

Air Microbiology↗
PubMed1982

A 3' co-terminus of two early herpes simplex virus type 1 mRNAs.

A 3' co-terminus of two early herpes simplex virus type 1 mRNAs has been identified using the nuclease -S1 mapping procedure with cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region 0.56-0.60, are unspliced and are transcribed rightwards on the prototype genome orientation. The position of their 3' ends has been located on the virus DNA sequence and lies downstream from the polyadenylation signal 5'-AATAAA-3'. This hexanucleotide sequence also was present in the complementary DNA strand and was shown to be the polyadenylation signal for a leftwards-transcribed late mRNA. The abundance within the cytoplasm of the 5.0 kb and 1.2 kb mRNAs was investigated. Results indicated that these mRNAs were regulated in concert. It is suggested that sequences at the 3' co-terminus may be involved in their regulation.

Base Sequence↗
PubMed1981

Cast bracing of femoral shaft fractures in children: a preliminary report.

This paper reviews the use of cast bracing in fractures of the femoral shaft in children between 1977 at the Royal Aberdeen Children's Hospital. Thirteen children have been treated by cast bracing. All children have achieved a full functional recovery. The average time to cast bracing was 3 weeks, and the average time to discharge was 4 weeks. Thus treated, these children were discharged 4 weeks earlier than those treated by conventional measures. It was found that the technique of cast bracing was applicable to children with no adverse complications.

Braces↗
PubMed1981

A biomechanical study of cast-brace treatment of femoral shaft fractures.

The off-loading characteristics of the cast-braces of 30 patients with fractures of the shaft of the femur have been investigated, during axial loading, using strain-gauge transducers. These were applied at the level of the fracture, where the cast was circumferentially split, and to the hinges of the brace at the knee. They measured the load transferred between the two portions of the thigh cast, and between the thigh cast as a whole and the below-knee cast; by subtraction from the total load on the limb, the skeletal force at the fracture level and at the knee could be calculated. In all patients there was an increase in the fracture load as union progressed which was thought to be due to physiological feedback mechanism from the fracture site. The load carried by the two portions of the thigh cast and by the thigh cast as a whole was proportionately high at first and stabilised at an average of 35 per cent of body weight.

Adolescent↗
PubMed1979

Orientation of herpes simplex virus type 1 immediate early mRNA's.

We have determined the orientation of 4 immediate early (IE) mRNA's on the herpes simplex virus type 1 genome by mapping cDNA's complementary to the 3'-termini of messages. These IE mRNA's are transcribed by a pre-existing cell RNA polymerase, and we propose a model which allows their synthesis from a circular template using a single virus promoter region. The promoter region, which is located in the two repetitive DNA regions which flank the short unique region of the virus genome, may serve to initiate bidirectional transcription of these IE mRNA's.

Base Sequence↗