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Biomedical subjects

E Goldman

Publications and source records attributed to E Goldman.

At least 19 recordsLinked to original sources
PubMed2001

Absence of effect of varying Thr-Leu codon pairs on protein synthesis in a T7 system.

The over-represented threonine-leucine (Thr-Leu) codon pair ACG CUG has been previously reported to be inhibitory to translation compared to the synonymous under-represented Thr-Leu codon pair ACC CUG, in an E. coli system in which the codon pairs were located either 3 and 4, or 6 and 7, or 9 and 10 codons downstream from the initiating codon for the message [Irwin, B., Heck, J. D., and Hatfield, G. W. (1995) J. Biol. Chem. 270, 22801-22806]. In the work reported here, these synonymous codon pairs were tested in a T7 system, with the codon pairs located either 14 and 15, or 6 and 7 codons downstream from the AUG start codon. In contrast to the reported findings in the E. coli system, there was no difference found in translation between mRNAs containing the respective codon pairs in the T7 system. The reasons for the different findings remain unclear, but presumably are a consequence of differences between the E. coli and T7 systems used to assay gene expression. Nevertheless, as a result of this work, it appears that the effect of varying codon pairs reported in the E. coli system is not due to a difference in translational step times through the respective codon pairs, as previously proposed.

Bacteriophage T7↗
PubMed2001

Reversal of inhibition by the T7 concatemer junction sequence on expression from a downstream T7 promoter.

We have previously reported that placement of the phage T7 concatemer junction (CJ) just upstream of another gene on a plasmid in a T7 system proved to be inhibitory to expression of the downstream gene. We had hypothesized that the inhibition was a result of a readthrough transcript of the CJ element interacting with the translation start region of the downstream gene; also that in the absence of a T7 termination signal, transcription continued around the plasmid multiple times ("rolling circle" transcription), always juxtaposing the inhibitory CJ sequence proximal to the downstream gene mRNA. Two strong predictions were made from this model: 1) that introduction of a spacer sequence between the CJ element and the downstream gene should alleviate the inhibition, and 2) that reintroduction of a T7 transcription terminator should prevent rolling circle transcription, thereby reversing the inhibition by allowing some transcripts to be generated originating from the downstream promoter that did not contain the inhibitory CJ element upstream. We report here that both of these predictions have been fulfilled. However, the reversal of inhibition was only partial in the construct where the T7 terminator was reintroduced, indicating that there remains a residual inhibitory effect of the CJ element on expression of the downstream gene. A possible explanation is that the CJ element, acting as a pause site for transcription, blocks access to the downstream T7 promoter, thereby reducing transcription from that promoter. If this explanation is correct, steric hindrance of transcription starts resulting from an upstream RNA polymerase pause site may represent a previously unrecognized mechanism of transcriptional control.

Bacteriophage T7↗
PubMed2000

Reproductive choices of women in families with haemophilia.

To assess women's experiences in pregnancy and attitudes towards their reproductive choices, a structured questionnaire was sent to all obligate and potential carriers of haemophilia (A and B), aged 14-60 years, registered with our haemophilia centre. One hundred and ninety-seven of 545 (36%) returned completed questionnaires. Clinical details, including type and severity of the disease in the family and results of DNA analysis for carrier detection, were obtained from patient notes. One hundred and sixty women had been pregnant at least once, of whom 36 (23%) had received a prenatal diagnostic test. Of the 41 women who had pregnancy terminations, haemophilia was the main reason in only 11 (27%) women. This decision was affected by the woman's religion and results of DNA studies. Living close to a haemophilia centre, proper counselling at the centre and awareness of the availability of prenatal diagnostic tests influenced the women's decision to become pregnant in 14% and 10% of first and subsequent pregnancies, respectively. These factors were considered more frequently in women with severe haemophilia in the family (P = 0.002) and in confirmed carriers of haemophilia (P = 0.04). When women made a conscious decision not to have children, the reasons were fear of passing haemophilia onto their child (44%), previous experience with haemophilia (6%) and the stress of going through prenatal tests (7%). Severity of the disease in the family, haemophilia diagnosis, results of DNA studies, religion and year of birth had no effect on this decision. Our data indicate that haemophilia and related factors in the family have an influence on women's reproductive choices.

Abortion, Induced↗
PubMed2000

Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display.

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.

Amino Acid Sequence↗
PubMed1999

Modified nucleosides in the first positions of the anticodons of tRNA(Leu)4 and tRNA(Leu)5 from Escherichia coli.

Minor leucine tRNA species, tRNA(Leu)4 and tRNA(Leu)5, from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman, E., Holmes, W. M., and Hatfield, G. W. (1979) J. Mol. Biol. 129, 567-585]. In the present study, these two tRNA(Leu) species were purified from E. coli A19, and the nucleotide sequences were determined by a post-labeling method. tRNA(Leu)5 was found to correspond to the tRNA gene reported as su degrees6 tRNA [Yoshimura, M., Inokuchi, H., and Ozeki, H. (1984) J. Mol. Biol. 177, 627-644]. The first letter of the anticodon was identified to be 2'-O-methylcytidine (Cm). tRNA(Leu)4 was identified as the minor leucine tRNA that has been sequenced previously (tRNA(Leu)UUR) [Yamaizumi, Z., Kuchino, Y., Harada, F., Nishimura, S., and McCloskey, J. A. (1980) J. Biol. Chem. 255, 2220-2225]. There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA(Leu)4. Nucleoside N* was isolated to homogeneity (1 A260 unit). By 1H NMR spectroscopy, nucleoside N was found to be a 2'-O-methyluridine derivative with a substituent having a -CH2NH2+CH2COO- moiety in position 5 of the uracil ring. On the basis of these NMR analyses together with mass spectrometry, the chemical structure of nucleoside N* was determined as 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um). Nucleoside N* was thus found to be a novel type of naturally occurring modified uridine. Because of the conformational rigidity of Cm and cmnm5Um in the first position of the anticodon, these tRNA(Leu) species recognize the leucine codons UUA++ and UUG correctly, but never recognize the phenylalanine codons UUU and UUC.

Anticodon↗
PubMed1999

Influence of atracurium on the diaphragm mean action potential conduction velocity in canines.

BACKGROUND: It has been shown that progressive neuromuscular blockade (NMB) affects the electromyogram power spectrum and compound muscle action potential duration in skeletal muscle. These measures are linked to the mean muscle action potential conduction velocity (APCV), but no studies have confirmed a relation between the mean APCV and NMB. The aim of this study was to determine whether diaphragm mean APCV is affected by NMB. METHODS: The effects of NMB on diaphragm mean APCV were evaluated in five mongrel dogs. Progressive NMB was induced by slow intravenous infusion of atracurium. During spontaneous breathing, the diaphragm mean APCV was determined by electromyogram signals, in the time and frequency domains. The magnitude of NMB was quantified by the amplitude of the compound muscle action potential and by changes in muscle shortening during supramaximal stimulation of the phrenic nerve. RESULTS: Progressive NMB was associated with a decrease in diaphragm mean APCV. At approximately 70% reduction in the compound muscle action potential amplitude, diaphragm mean APCV had decreased more than 20%. Recovery after NMB was characterized by a restoration of the mean APCV to control values. CONCLUSION: This study shows that progressive NMB paralyzes motor units within the diaphragm in an orderly manner, and the blockade first affects muscle fibers with high APCV before it affects fibers with lower APCV.

Action Potentials↗
PubMed1999

The T7 concatemer junction sequence interferes with expression from a downstream T7 promoter in vivo.

A recently described new signal for transcription termination in vitro by T7 RNA polymerase has now been tested in vivo. This signal, identified during transcription of the cloned human preproparathyroid hormone (PTH) gene, is also found in the phage T7 genome, at the concatemer junction (CJ). We introduced the 17-bp concatemer junction sequence at the ends of a test gene and control gene (both derived from T7 gene 9) in a T7 vector previously used to study effects of rare codons on expression. The CJ elements replaced the original vector's RNase III processing sites, and a new T7 promoter was also introduced to drive the downstream (control) gene. We assayed for test and control gene mRNA and protein by direct labeling with [32P]phosphate and [35S]methionine. The altered vector with CJ sequences (pCT1.1) expressed the upstream test gene, but showed poor expression of the downstream control gene. No discrete T7 mRNA bands could be discerned by direct labeling with 32P. A precursor vector with only the control gene in single copy expressed the protein much better, suggesting that the inhibition of control gene expression in pCT1.1 was a result of the upstream CJ element at the 3' end of the test gene. RT-PCR experiments were consistent with readthrough and possibly pausing at CJ. An RNA folding program predicts a highly stable secondary structure between the upstream CJ element and the control gene's translation start signals. These data support an interpretation that the CJ element is ineffective as a T7 transcription terminator in vivo in this vector, and that structure of the readthrough transcript blocks ribosome access to the downstream translation start. The readthrough transcripts are also likely to be less stable than properly terminated or processed T7 mRNA, because levels of test protein expression in pCT1.1 were reduced compared to original vector, and basal expression was negligible, while the original codon test vector shows substantial basal expression.

Bacteriophage T7↗
PubMed1997

Messenger RNA release from ribosomes during 5'-translational blockage by consecutive low-usage arginine but not leucine codons in Escherichia coli.

In '5'-translational blockage', significantly reduced yields of proteins are synthesized in Escherichia coli when consecutive low-usage codons are inserted near translation starts of messages (with reduced or no effect when these same codons are inserted downstream). We tested the hypothesis that ribosomes encountering these low-usage codons near the translation start prematurely release the mRNA. RNA from polysome gradients was fractionated into pools of polysomes and monosomes and a ribosome-free pool. New hybridization probes, called 'molecular beacons', and standard slot blots were used to detect test messages containing either consecutive low-usage AGG (arginine) or synonymous high-usage CGU insertions near the 5' end. The results show an approximately twofold increase in the ratio of free to bound mRNA when the low-usage codons were present in the message compared with when high-usage codons were present. In contrast, there was no difference in the ratio of free to bound mRNA when consecutive low-usage CUA or high-usage CUG (leucine) codons were inserted or when the arginine codons were inserted near the 3' end. These data indicate that at least some mRNA is released from ribosomes during 5'-translational blockage by arginine but not leucine codons, and they support proposals that premature termination of translation can occur in some conditions in vivo in the absence of a stop codon.

Arginine↗
PubMed1997

Use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to resolve mRNA and its protein product in one gel.

We demonstrate that it is possible to simultaneously resolve both an mRNA and its protein product by electrophoresis in a single SDS-polyacrylamide gel by using double labeling with [32P]H3PO4 and [35S]methionine, and an elongated 5% stacking gel atop the 10% resolving gel. The mRNA is resolved in the 5% gel; the protein, as expected, resolves in the 10% gel. Using a T7 expression system, we show that putative mRNA bands in the 5% gel are: 1) labeled only with 32P and not with 35S; 2) inducible with isopropylthiogalactopyranoside (needed to induce a T7 RNA polymerase gene under control of a lac promoter); 3) synthesized in the presence of rifampicin (T7 RNA polymerase is not inhibited by rifampicin); 4) degraded by base or RNase treatment; and 5) are largely resistant to DNase treatment. The mRNA bands were also evident in samples not treated with rifampicin. We used this technique to confirm previously published results that inhibition of expression by consecutive low-usage AGG arginine codons inserted near the 5' end of a test message in Escherichia coli is at the level of translation.

Autoradiography↗
PubMed1997

The obstetric experience of carriers of haemophilia.

OBJECTIVE: To review the obstetric problems, pregnancy outcome and management of carriers of haemophilia. DESIGN: Retrospective review of haemophilia and maternity unit records. SETTING: Haemophilia Comprehensive Care Centre. PARTICIPANTS: Thirty-two carriers of haemophilia (24 haemophilia A, eight haemophilia B) who had their obstetric care at the Royal Free Hospital over a 10-year period (1985-1995). MAIN OUTCOME MEASURES: Uptake and results of prenatal diagnosis, changes in factor levels during pregnancy, effect of knowledge of fetal gender on obstetric management and neonatal outcome, and maternal haemorrhagic complications. RESULTS: There were 82 pregnancies and 32 resulted in miscarriage or social abortion. The option of prenatal diagnosis was taken up in only 35% (17/48) of pregnancies. There were five affected male fetuses diagnosed prenatally but only three women opted for termination of the pregnancy. Knowledge of fetal gender was unavailable to the attending obstetrician in 46% (21/46) of pregnancies. A fetal scalp electrode was applied in eight, fetal blood sampling was performed in four, and ventouse delivery was conducted in one of these pregnancies. No adverse effects were reported from the first two procedures, but the ventouse delivery was associated with a huge cephalhaematoma requiring blood transfusion. On the other hand, in five cases fetal blood sampling was withheld because fetal gender was unknown. Four of the eight caesarean sections performed might have been avoided if the gender had been known. The incidence of primary and secondary postpartum haemorrhage was high, 22% (including two cases with massive haemorrhage) and 11%, respectively. CONCLUSION: Carriers of haemophilia A and B require special obstetric care with close liaison with the haemophilia centre, and management guidelines should be available and observed. Knowledge of fetal gender is very valuable for management in labour and should be determined antenatally even if the mother declines prenatal diagnosis.

Abortion, Induced↗
PubMed1996

Analysis of diaphragm EMG signals: comparison of gating vs. subtraction for removal of ECG contamination.

The diaphragm electromyogram (EMGdi) conveys information relating to the mechanisms of respiration; however, electrocardiogram (ECG) contamination can compromise the accuracy of data derived from this signal. We examine the EMGdi recorded from anesthetized spontaneously breathing dogs via implanted electrodes to assess the extent of the error introduced by the ECG contamination and the effectiveness of ECG gating in reducing this error. Because ECG subtraction has been shown to generate accurate results for such applications, it is used as the gold standard. Analysis of variance methods are employed to compare results derived from the EMGdi data after ECG subtraction with corresponding results derived from the original data and from the data after ECG gating. Estimates of EMGdi variables obtained by using subtraction and gating techniques were not significantly different, indicating that gating can be employed on these signals to reduce ECG contamination without affecting the accuracy of the derived data. Results also show that at EMG-to-ECG power ratios > 13.3 dB, ECG contamination does not significantly affect estimates of the EMGdi variables.

Animals↗
PubMed1996

[Mitomycin C for the treatment of superficial tumors of the bladder (Tis-Ta-T1) with one or more risk factors. Argentinian Cooperative Group of Oncourology (GCAO)].

OBJECTIVES: To determine the efficacy and toxicity of intravesical mitomycin C in superficial bladder tumors (Tis Ta T1) completely resected with one or more risk factors. (Protocol U. 01/90). METHODS: The patients received six instillations weekly of 40 mg mitomycin C within 15 days post-TUR of a superficial bladder tumor with one or more risk factors: histological grade 3, tumor size more than 3 cm and/or multicentric lesion. The patients were evaluated by cystoscopy every three months for the first two years and every six months thereafter. Toxicity was evaluated according to Miller's score. Tumor recurrence, disease-free interval and survival were analyzed. RESULTS: 126 patients were entered into the study; of these, 110 were evaluable. At 18 months mean follow-up (range 6-36 months), 77 patients (70%) remain disease-free; the mean time to recurrence was 13.8 months. There were no differences between patients with one, two or three risk factors or those who received or did not receive previous treatments. The patients tolerated the treatment well; there were no dropouts or systemic toxicity. CONCLUSIONS: 1. At 18 months mean follow-up, 77 of 110 patients (70%) remain disease-free; 2. The mean time to recurrence was 13.8 months; 3. Local toxicity was minimal; 4. There were no dropouts due to toxicity; 5. Systemic toxicity was not observed.

Adult↗
PubMed1995

Evidence that uncharged tRNA can inhibit a programmed translational frameshift in Escherichia coli.

In the modified release factor 2 (RF2) programmed translational frameshift (with a sense codon replacing the wild-type in-frame UGA codon at the shift site), ribosomes shift +1 into the reading frame for an out-of-frame reporter fused to the frameshift sequence. Partitioning of ribosomes between the out-of-frame shift and in-frame reading depends on the codon at the shift site and on the levels of tRNA decoding the in-frame codon. Overexpression of a tRNA species cognate to the in-frame codon at the shift site significantly reduces the frequency of frame-shifting, presumably by facilitating in-frame reading, which would reduce production of the out-of-frame reporter. However, since overexpression of a tRNA increases levels of both charged and uncharged tRNA, it is possible that uncharged cognate tRNA might be able to reduce the frequency of the frameshift, by entering the A site on the ribosome. To test this, we manipulated charged and uncharged tRNA levels in vivo, using the tryptophan analog tryptophan hydroxamate, which increases the proportion of uncharged tRNA(Trp) by competing with cognate amino acid tryptophan for tryptophanyl-tRNA synthetase, thereby reducing protein synthesis. We report here that a slight but reproducible reduction in the relative frequency of the frameshift is observed when tryptophan hydroxamate is added to cells containing the modified RF2 shift with UGG (Trp codon) at the shift site. When tRNA(Trp) is overexpressed from another plasmid, the shift frequency drops three- to fourfold, as expected, however, this reduction is still seen in the presence of the analog. Thus, under conditions when most of the tRNA(Trp) is apparently uncharged, excess tRNA(Trp) still causes a significant reduction in the frameshift when UGG is at the shift site, providing evidence that uncharged cognate tRNA also can inhibit this frameshift.

Amino Acyl-tRNA Synthetases↗