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Background: The physiological changes of pregnancy can increase the risk of peri-partum pulmonary aspiration. There is limited objective information regarding gastric volumes in pregnant patients. The aim of this cohort study was to characterise prospectively the range of gastric-fluid volume in term non-labouring pregnant patients compared with a historical cohort of non-pregnant females. Methods: Fasted non-labouring term pregnant patients scheduled for elective Caesarean delivery underwent a standardised gastric ultrasound examination. Gastric content was evaluated qualitatively (type of content), semiquantitatively (Perlas grades), and quantitatively (volume). The antral cross-sectional area and volume were compared with those of a retrospective cohort of non-pregnant females from the same institution. Descriptive statistics were used to describe the central tendency through mean and median values. Dispersion was evaluated with standard deviation and inter-quartile range, and the higher end of the distribution as 95th percentile. Results: Non-labouring pregnant (59) and non-pregnant (81) subjects were studied. The range of estimated total gastricfluid volume (P = 0.96) and volume per body weight (P = 0.78) was not significantly different between cohorts. An estimated volume of 115 ml (102-143) vs 136 ml (106-149) and volume per body weight of 1.4 ml kg(-1) (1.2-2.8) vs 2.0 ml kg(-1) (1.5-2.7) corresponded to the 95th percentile (95% confidence interval) values in the pregnant and non-pregnant cohort, respectively. Conclusions: Baseline gastric volume of non-labouring pregnant patients at term is not significantly different from that of non-pregnant females. This information will be helpful to interpreting findings of gastric point-of-care ultrasound in obstetric patients.

Contains fulltext : 229026.pdf (Publisher’s version ) (Closed access) Personalised risk assessment of the likelihood of pulmonary aspiration is recommended for pregnant women undergoing general anaesthesia and gastric point-of-care ultrasound (PoCUS) may help to achieve this. Traditionally, risk assessment is based upon adherence to fasting times, but gastric emptying may vary during pregnancy and surgery often needs to be expedited. We systematically reviewed the evidence for gastric PoCUS up to August 2018 in pregnant and postpartum women to determine whether it can identify and quantify stomach contents, provide aspiration risk assessment via qualitative or quantitative means, and determine how gastric emptying is affected by pregnancy. Twenty-two articles comprising 1050 participants were included and studies were classified by qualitative or quantitative findings. The evidence suggests that gastric PoCUS is a reliable and feasible method of imaging the stomach in pregnancy in clinical practice. Qualitative assessment via the Perlas grading system can provide rapid assessment of gastric volume states. If fluid is visible, identification of patients at high risk of pulmonary aspiration requires measurement of antral cross-sectional area. Cut-off values of 608mm(2) and 960mm(2) are recommended in the semi-recumbent and right lateral semi-recumbent positions, respectively. Validated methods to quantify stomach volumes are available, however their usefulness is currently restricted to research. Gastric PoCUS also provides evidence that gastric emptying of ingested food is delayed by term pregnancy, labour and during the early postpartum period. However, the passage of fluids through the stomach appears unaffected throughout the peripartum period.

ispartof: INDIAN JOURNAL OF ANAESTHESIA vol:63 issue:2 pages:160-161 ispartof: location:India status: published

Pulmonary aspiration of gastric content is a serious anaesthetic complication that can lead to significant morbidity and mortality. Aspiration risk assessment is usually based on fasting times. However, fasting guidelines do not apply to urgent or emergent situations and to patients with certain co-morbidities. Gastric content and volume assessment is a new point-of-care ultrasound application that can help determine aspiration risk. This systematic review summarizes the current literature on bedside ultrasound assessment of gastric content and volume relevant to anaesthesia practice. Seventeen articles were identified using predetermined criteria. Studies were classified into those describing the sonographic characteristics of different types of gastric content (empty, clear fluid, solid), and those describing methods for quantitative assessment of gastric volume. A possible algorithm for the clinical application of this new tool is proposed, and areas that require further research are highlighted.

Contains fulltext : 176961.pdf (Publisher’s version ) (Closed access) Background: Perioperative aspiration leads to significant morbidity and mortality. Point-of-care gastric ultrasound is an emerging tool to assess gastric content at the bedside. Methods: We performed a retrospective cohort study of baseline gastric content on fasted elective surgical patients. The primary outcome was the incidence of full stomach (solid content or >1.5 ml kg-1 of clear fluid). Secondary outcomes included: gastric volume distribution (entire cohort, each antral grade); the association between gastric fullness, fasting intervals, and co-morbidities; anaesthetic management changes and incidence of aspiration. Results: We identified 538 patients. Thirty-two patients (6.2%) presented with a full stomach. Nine of these (1.7%) had solid content and 23 (4.5%) had clear fluid >1.5 ml kg-1. An empty stomach was documented in 480 (89.8%) patients. The examination was inconclusive in the remaining 20 patients (5.0%). As expected, increasing antral grade was correlated with larger antral cross-sectional area and higher gastric volume (P

The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpathway. Although rad26 disruption mutants are defective in transcription-coupled repair, they are not UV sensitive. However, double mutants of RAD26 with the global genome repair determinants RAD7 and RAD16 appeared more UV sensitive than the single rad7 or rad16 mutants but not as sensitive as completely NER-deficient mutants. These findings unmask a role of RAD26 and transcription-coupled repair in UV survival, indicate that transcription-coupled repair and global genome repair are partially overlapping, and provide evidence for a residual NER modality in the double mutants. Analysis of dimer removal from the active RPB2 gene in the rad7/16 rad26 double mutants revealed (i) a contribution of the global genome repair factors Rad7p and Rad16p to repair of the transcribed strand, confirming the partial overlap between both NER subpathways, and (ii) residual repair specifically of the transcribed strand. To investigate the transcription dependence of this repair activity, strand-specific repair of the inducible GAL7 gene was investigated. The template strand of this gene was repaired only under induced conditions, pointing to a role for transcription in the residual repair in the double mutants and suggesting that transcription-coupled repair can to some extent operate independently from Rad26p. Our findings also indicate locus heterogeneity for the dependence of transcription-coupled repair on RAD26.

The Gin invertase of bacteriophage Mu mediates recombination between two inverted gix sites. Recombination requires the presence of a second protein, Fis, which binds to an enhancer sequence. We have isolated 24 different mutants of Gin that are impaired in DNA inversion but proficient in DNA binding. Six of these mutants could be suppressed for inversion by introduction of a second mutation, which when present in the wild-type gin gene causes a Fis-independent phenotype. Only one of the six resulting double mutants shows an inversion efficiency which is comparable to that of the wild-type Gin and which is independent of Fis. The corresponding mutation, M to I at position 108 (M108I), is located in a putative alpha-helical structure, which in the homologous gamma delta resolvase has been implicated in dimerization. The properties of the M108I mutant suggest that in Gin this dimerization helix might also be the target for Fis interaction. The five other mutants that show a restored inversion after introduction of a Fis-independent mutation appear to be completely dependent on Fis for this inversion. The corresponding mutations are located in different domains of the protein. The properties of these mutants in connection with the role of Fis in inversion will be discussed.

Transcription-coupled repair (TCR) is a universal sub-pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription-blocking DNA lesions and permits rapid resumption of the vital process of transcription. A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS). Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B. Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26. A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function. In analogy with CS, preferential repair of UV-induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired. Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV-, cisPt- or X-ray sensitivity, explaining why it was not isolated as a mutant before. Recovery of growth after UV exposure was somewhat delayed in rad26. These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity.

The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs.

The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repair is influenced by the nature of the lesion, the local DNA sequence, and/or the topology of the DNA. To get a better understanding of the aspects of this multistep repair reaction that determine the effectiveness of repair, we compared the incision efficiencies of linear DNA fragments containing either a site-specific cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2)]] or a cis- Pt(NH3)2[d(GpCpG)-N7(1),-N7(3)]] adduct. Overall the DNA with the cis-PtGG adduct was incised about 3.5 times more efficiently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preincision complex formation for both lesions was similar and high in relation to the incision. DNase I footprints, however, showed that the local structure of the two preincision complexes is different. An assay was developed to measure the binding of UvrC to the preincision complexes and it was found that the binding rate of UvrC to the more slowly incised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative difference in preincision complex structures. For both lesions the binding of UvrC to the preincision complex was fast compared to the kinetics of actual incision. Apparently, direct incision of cisplatin damage requires an additional conformational change after the binding of UvrC.

Epidermal growth factor (EGF) enhances the expression of the keratinocyte terminal differentiation marker SPRR2A, when added to monolayers of basal keratinocytes, induced to stratify by increasing the extracellular calcium concentration. A similar stimulation is found during suspension-induced differentiation in methylcellulose. This effect, which is observed after several hours of EGF addition, is restricted to terminally differentiating keratinocytes and is dependent on PKC signaling. EGF also transiently activates the Ras signaling pathway, with a maximum induction after 10 min (Medema et al., 1994, Mol. Cell. Biol. 14, 7078-7085). The cellular effects of activated Ras were determined by transient transfection of Ha-ras(Leu-61) into normal human keratinocytes. Activated Ras completely inhibited PKC-mediated expression of SPRR2A. This inhibition is mediated via c-Jun as it is reversed by a dominant-negative c-Jun mutant (cJunDelta6/194) and c-Jun can substitute for activated Ras. The inhibitory effect is targeted to a 150-bp minimal promoter region, which is essential and sufficient for SPRR2A expression during keratinocyte terminal differentiation. This indicates that the Ras and PKC pathways, which both can be triggered by EGF, although at different time points, have opposite effects on SPRR2A gene expression.

Nucleotide excision repair (NER) is the main process used by cells to remove bulky lesions from DNA (reviewed in Friedberg et al. 1995). The molecular details of its mechanism have become increasingly clear during the past few years, and NER appears to be remarkably conserved during evolution from yeast to man. The role of the proteins involved has been studied at the biochemical level and eukaryotic NER has been reconstituted in highly purified in vitro systems (Aboussekhra et al. 1995; Guzder et al. 1995; Mu et al. 1995). In yeast, the Rad1/10 and Rad2 endonucleases, the Rad4/23 complex, the trimeric Rpa protein, the Rad14 DNA-damage recognizing protein, and the transcription/NER complex TFIIH are all necessary and sufficient for incision of damaged DNA (Guzder et al. 1995). These proteins will be referred to as the core NER proteins. Although all nucleotide excision repair appears to require at least this set of proteins, additional factors may be involved in vivo. The kinetics of removal of cyclobutane pyrimidine dimers (CPDs), the major lesions induced by UV-light, are not equal for all DNA in the cell: transcribed strands are repaired faster than non-transcribed DNA. We will discuss recent advances that give insight into the mechanism behind this observation, termed differential repair, leading to the hypothesis that differential repair is due to the operation of two subpathways of NER: transcriptioncoupled and global genome repair. The genes that are implicated in these subpathways in yeast and mammalian cells will be discussed.

The small proline rich protein (SPRR) genes constitute a family of conserved genes which are part of the human epidermal differentiation complex (EDC) on chromosome 1q21 and code for precursor proteins of the cornified cell envelope. The expression of these genes is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. Hero we show that cultured cell lines derived from squamous cell carcinoma (SCC) show significantly lower levels of SPRR expression than normal human keratinocytes. However, the residual SPRR expression in SCC lines appears to be both gene and cell line specific. Expression of SPRR2 appears to correlate well with the residual ability of these cells to differentiate. However, the kinetics of SPRR2 expression, following treatment with calcium, an inducer of keratinocyte differentiation, are typical for each cell line and differ substantially from the ones found in normal cells. In most cell lines a rapid transient expression of SPRR2 contrasts with a slow induction leading to a high sustained level of expression in normal cells. This pattern of expression is typical for SPRR2 and not observed for the other SPRR genes or involucrin. Our analysis indicates that the expression of various keratinocyte terminal differentiation markers, even when involved in the same biological process (cornification), can be differentially affected by carcinogenic transformation.

To evaluate the diagnostic and therapeutic implications of transesophageal echocardiography (TEE) in intensive care patients.Comparative study.A 10-bed general intensive care unit.Between 1 January 1992 and 31 May 1993, 61 patients prospectively identified with shock (n = 14), severe, unexplained hypoxemia (Partial pressure of oxygen in arterial blood/fractional inspired oxygen200) (n = 31), or suspected endocarditis (n = 16) underwent a TEE examination to supplement transthoracic echocardiography (TTE) examination.The results of each TEE examination were compared with the clinical findings and TTE data. TEE examinations were classified as follows: 0, TEE results were similar to TTE results; 00, TEE examination resulted in exclusion of suspected abnormalities; 1, TEE revealed a new but minor diagnosis compared to the TTE diagnosis; 2, TEE revealed a new major diagnosis not requiring a change of treatment; 3, TEE results revealed a new major diagnosis requiring an immediate change of treatment.Intraobserver reliability of the TEE classification was confirmed by a 100% concordance and interobserver reliability was evaluated as an 84% concordance. Results of the TEE classification were: class 0, n = 21 (34%); class 00, n = 13 (21%); class 1, n = 7 (12%); class 2, n = 8 (13%); class 3, n = 12 (20%). Therapeutic implications of TEE in class 3 patients were cardiac surgery in 5 patients (2 cases of acute mitral regurgitation, 2 valvular abscesses, and 1 hematoma compressing the left atrium), discontinuation of positive end-expiratory pressure in 1 ventilated patient with an atrial septal defect, weaning off mechanical ventilation in 1 patient with an atrial septal defect, prescription of antimicrobial therapy in 3 patients with endocarditis, and prescription of anticoagulant therapy in 2 patients with left atrial thrombus. No difficulty inserting the transducer was observed in any of the 61 patients studied. The only noteworthy complication was a case of spontaneously resolving atrial fibrillation.TEE is safe and well tolerated and is useful in the management of patients in the intensive care unit with shock, unexplained and severe hypoxemia, or suspected endocarditis when TTE is inconclusive.

The RAD23 gene of Saccharomyces cerevisiae is involved in nucleotide excision repair (NER) and mutations in this gene confer a moderate sensitivity to UV irradiation. However, no repair of either cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, the major types of lesions formed upon UV irradiation, was detectable during the first 4 h post UV irradiation in a rad23 mutant. rad23, like the rad7 and rad16 mutants, is not as UV sensitive as completely NER-deficient mutants. The rad7 and rad16 mutants are only partly defective in NER: non-transcribed strands are completely refractory to repair while transcription-coupled repair is not affected. To investigate whether the rad23 mutant has similar strand-specific repair characteristics we analyzed gene-specific CPD removal from several loci using strand-specific probes but did not detect any repair. The moderate UV sensitivity of rad23 mutants as compared to completely NER-deficient mutants is therefore not due to gene- or strand-specific removal of lesions, indicating that rad23 mutants do not have a similar repair defect as rad7 or rad16 mutants, but are presumably defective in general NER. The rad23 mutation does not suppress the high UV sensitivity of completely NER-deficient rad1 or rad14 strains. This demonstrates that the relatively high survival of rad23 mutants in not due to an increased tolerance for the lesions that seem to persist in the genome but rather requires some NER function.

The toxic effect and the mutagenicity of two differentially repaired site-specific cis-diamminedichloroplatinum(II) (cis-DDP) lesions were investigated. Detailed analysis of the UvrABC-dependent repair of the two lesions in vitro showed a more efficient repair of the cis-Pt.GG adduct compared to that of the cis-Pt.GCG adduct (Visse et al., 1994). Furthermore, previously, a dependency of cis-DDP mutagenesis on UvrA and UvrB, but not on UvrC was found (Brouwer et al., 1988). To possibly relate survival and mutagenesis to repair, plasmids containing the same site-specific cis-DDP lesions as those that were used in the detailed repair studies were transformed into Escherichia coli. The results indicate that both lesions are very efficiently bypassed in vivo. Mutation analysis was performed using a denaturing gradient gel electrophoresis technique, which allows identification of mutations without previous selection. Although the cis-Pt.GG adduct is in vitro more efficiently repaired than the cis-Pt.GCG adduct, it appeared to be more mutagenic. We present a model in which this result is related to the previously observed dependency of the mutagenicity of cis-DDP lesions on the Uvr A and B proteins.

Sudden non-traumatic death in young people (< 30 years old) has been discussed both in systematic studies and anecdotal reports. After presenting three remarkable cases, a global survey of the incidence with special reference to the Belgian CPCR database, ethiopathogenesis and prognosis of sudden non-traumatic death in this specific age group is given. The atherosclerotic coronary artery disease (CAD) related and especially the non-CAD related causes are extensively discussed as well as the role of intoxication and increasing drug and substance abuse. After conventional cardiopulmonary resuscitation with restoration of spontaneous circulation and a favourable neurological outcome, a thorough search for the underlying disease is mandatory. The involvement of drugs or other toxins has to be excluded in the first place. Apart from transoesophageal echocardiography and coronary angiography, electrophysiological testing, serological exams, myocardial biopsy and magnetic resonance imaging should be considered.

The influence of the genomic position of a gene on UV-induced mutations was studied in the endogenous hprt gene in human lymphoblastoid TK6 cells and in cell lines derived from TK6 each containing a single copy of a hamster hprt cDNA gene integrated on a retroviral vector in different positions of the human genome. Previous studies showed that the genomic sequences surrounding the integration site influence spontaneous mutagenesis, resulting in a 10-fold difference in mutation rates among the hprt cDNA genes. Here we demonstrate that the genomic positions of three integrated hprt cDNA genes do not influence UV-induced mutagenesis. The mutability by UV irradiation in these cell lines is approximately the same (16.0 x 10(-6) per J/m2). The nature of the UV-induced mutations determined in two of the cell lines containing the integrated hprt cDNA gene (approximately 30 mutants each) was also found not to be different. The endogenous hprt gene in the parental TK6 cells exhibits a significantly lower mutability (2.1 x 10(-6) per J/m2) than the cDNA genes, but the spectrum is very similar. The spectrum in TK6 shows no influence of strand-specific repair and resembles most closely the spectrum obtained by McGregor et al. after irradiation of human cells synchronized in S-phase. This suggests that mutations arising in cells that are in S-phase at the time of irradiation constitute the majority of the mutants in an asynchronous TK6 cell population. We hypothesize that repair in the endogenous hprt gene in TK6 cells is very efficient, removing virtually all lesions before replication takes place except in cells that were in S-phase at the time of irradiation when there is not enough time for repair. Furthermore we suggest that the higher mutability of the integrated hprt cDNA genes compared with the endogenous gene is caused by a less efficient repair in the cDNA genes.

Summary In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3–80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site leads to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.

Escherichia coli contains pyrroloquinoline quinone-dependent glucose dehydrogenase. We cloned and sequenced the gene (gcd) encoding this enzyme and showed that the derived amino acid sequence is highly homologous to that of the gdhA gene product of Acinetobacter calcoaceticus. Stretches of homology also exist between the amino acid sequence of E. coli glucose dehydrogenase and other pyrroloquinoline quinone-dependent dehydrogenases from several bacterial species. The position of gcd on the chromosomal map of E. coli was determined to be at 3.1 min.

The Gin product catalyzes an inversion of 3,000 base pairs of DNA in the genome of bacteriophage Mu. The orientation of the invertible of G-region determines the host range of the phage. Gin- mutants are complemented by a host function in strain HB101 and several other Escherichia coli K-12 strains. At least three clones in the E. coli gene bank described previously (L. Clarke and J. Carbon, Cell 9:91-99, 1976) contained the gin complementing function. This function, which we named pin, catalyzes an inversion of 1,800 base pairs in the adjacent DNA. The invertible region, named the P-region, together with pin, was further subcloned on pBR322. Conjugation and transduction experiments mapped the pin gene between the genes purB and fabD near position 25 on the E. coli chromosome. Also situated in this region is e14, a cryptic, UV- excisable , genetic element (A. Greener and C.W. Hill, J. Bacteriol . 144:312-321, 1980). We demonstrated that pin and the P-region are part of e 14. The e 14 element was cloned on pBR322 by genetic manipulation techniques in vivo. It has the properties of a defective prophage containing integration and excision functions and a SOS-sensitive repressor.

Recently, two groups of cDNA clones have been isolated from human epidermal keratinocytes; the clones correspond to genes whose expression is stimulated by exposure of the cells to UV light or treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate (T. Kartasova and P. van de Putte, Mol. Cell. Biol. 8:2195-2203, 1988). The proteins predicted by the nucleotide sequence of both groups of cDNAs are small (8 to 10 kilodaltons), are exceptionally rich in proline, glutamine, and cysteine, and contain repeating elements with a common sequence, PK PEPC. These proteins were designated sprI and sprII (small, proline rich). Here we describe the characterization of the sprIa protein, which is encoded by one of the group 1 cDNAs. The expression of this protein during keratinocyte differentiation in vitro and the distribution of the sprIa protein in some human tissues was studied by using a specific rabbit antiserum directed against a synthetic polypeptide corresponding to the 30 amino acids of the C-terminal region of the sprIa gene product. The results indicate that the expression of the sprIa protein is stimulated during keratinocyte differentiation both in vitro and in vivo.

The Ner protein of bacteriophage Mu negatively regulates transcription initiated at the early promoter and at the major repressor promoter. The construction and isolation of a Ner- mutant of Mu is described. Ner is an essential function for Mu, because the mutant phage only forms plaques when complemented for Ner. Mutations in the repressor protein did not abolish the need for Ner. However, when transcription of the repressor gene c was blocked by deleting the major repressor promoter, Ner was no longer essential for normal Mu development.

In an approach to study effects of UV light on gene expression in human epidermal keratinocytes, a cDNA library was constructed from poly(A)RNA isolated after UV irradiation from cultured keratinocytes. The cDNA library was differentially screened with labelled cDNA probes synthesized on poly(A)RNA isolated from UV irradiated or nonirradiated keratinocytes. Forty clones were selected and subjected to further analysis, 31 of them are described in this report. Whereas total mRNA synthesis is reduced after UV irradiation or treatment with 4-NQO Northern blot analysis revealed that there is an at least relative increase in the level of mRNAs corresponding to the majority of the isolated cDNA clones. Among these 15 were identified as corresponding to mRNAs for 50K and 56K keratins and for 50K- and 46K-related keratin. In addition, clones were found corresponding to the proteinase inhibitor cystatin A and to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of keratinocytes with the tumor promoter TPA had no effect on the mRNA level for most of the clones except those corresponding to keratins. Our results indicate that in keratinocytes UV irradiation leads to a relative increase in the level of some mRNAs.

Cell killing and mutation induction by cis- and trans-Pt(NH3)2Cl2 in Escherichia coli were examined by studying forward mutagenesis in the lacI gene in cells with different repair capacities. Survival experiments showed that repair-proficient cells were slightly more sensitive for the cis isomer than for the trans isomer, whereas repair-deficient RecA and UvrB cells were extremely sensitive only for the cis compound. cis-Pt(NH3)2Cl2 induced mutagenesis in both wild-type cells and RecA cells but not in UvrB cells; whereas no detectable mutagenesis was induced by treatment with the trans compound. Examination of the nature of the mutations induced by cis-Pt(NH3)2Cl2, by using the LacI system, revealed that base-pair substitutions leading to nonsense mutants are only induced in wild-type cells, suggesting that the intact products of both the uvrB and the recA gene are necessary for the repair responsible for this type of mutagenesis. Investigation of the nonsense mutants reveals that 70% of these mutations result from GC leads to TA or GC leads to AT substitutions at sites where the guanine is part of a GAG or GCG sequence. These results are discussed in relation to existing theories on the interaction between Pt compounds and DNA. A model for Pt--DNA adducts, leading to base-pair substitutions, is proposed.

Inversion of the G segment in the DNA of Escherichia coli phage Mu depends on the Mu Gin protein and alters the host range of the phage. The frequency of the inversion reaction is low both in the lysogenic state and during lytic growth. A sensitive assay was developed to detect low levels of G inversion: the E. coli lac operon was inserted within the invertible G segment in such a way that the lac operon was expressed only by G(-) clones. As a result Gin-catalyzed inversion from G(+) to G(-) can be monitored as a lactose-negative to lactose-utilizing switch. Using a crude extract from a Gin-overproducing strain and this assay plasmid, we could detect a low level of G inversion in vitro (1% in 30 min). The reaction depends on Mg2+ and a supercoiled substrate. Under optimized reaction conditions over 15% of the plasmids had the G segment inverted after incubation with Gin in vitro. The inversion was then visualized by agarose gel analysis of plasmid DNA digested by restriction endonucleases. The Gin protein retains its catalytic properties upon partial purification. The mechanism of this genetic switch can now be studied in vitro.

Cell killing and mutation induction in the lacI gene of Escherichia coli by cis-Pt(NH3)2Cl2 were studied in cells with different repair capacities, with and without pKM101. The presence of the plasmid pKM101 made repair-proficient cells more susceptible to killing by cis-Pt(NH3)2Cl2 and strongly enhanced mutation induction by that compound. Both effects were shown to be dependent upon excision repair. Characterization of the induced mutations in the lacI gene after cis-Pt(NH3)2Cl2 treatment of E. coli cells, by the LacI system, revealed that the mutagenic specificity of the Pt compound was strongly influenced by the presence of the pKM101 plasmid. With pKM101, 23% of the induced amber and ochre mutations resulted from substitutions at AT base pairs, whereas these mutations were hardly induced in cells without pKM101. These results suggest that pKM101-induced repair differs from normal SOS repair.

We cloned the gene coding for the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. This clone complements gdh mutations in A. calcoaceticus, Pseudomonas aeruginosa, and Escherichia coli. The gene codes for a protein with an Mr of 83,000. Evidence is presented for the presence of two different glucose dehydrogenase enzymes in A. calcoaceticus: a protein with an Mr of 83,000 and a dimer of two identical subunits with an Mr of 50,000.

The gene product of bacteriophage Mu gin catalyzes a 3,000-base-pair inversion in the DNA of the phage, thus changing its host range. In some strains of Escherichia coli there is a function that can complement Mu gin mutations. This function (pin) was cloned and shown to catalyze an inversion of 1,800 base pairs in the adjacent E. coli DNA (P region). pin- derivatives carry the P region frozen in the (+) or (-) orientation. The function of the switch is not yet clear. The sequences of gin and pin were determined; they exhibit 70% homology. The sequences around the recombination sites of Gin and Pin are also largely homologous; a consensus sequence is derived for the recombination sites of Gin and Pin, and of Hin in Salmonella typhimurium. The amino acid sequences of Gin, Pin, Hin, and TnpR are compared, and the evolutionary relationship between these prokaryotic site-specific recombination systems is discussed.

By conjugation between Hfr and F − strains, the location on the bacterial chromosome of various genes controlling sensitivity to ultraviolet irradiation was determined in Escherichia coli strain B, strain K12S and strain CR 34. On account of their colony-forming ability after ultraviolet irradiation, the radiation sensitive mutants bearing the various markers studied appeared to fall into two classes: (a) extremely radiation-sensitive mutants hcr − , dar 1 − , dar 5 − and dar 6 − ; and (b) strains B s −2 , dar 3 − , dar 3 − and dar 4 − with a radiation sensitivity intermediate between that of the extremely sensitive strains and that of their corresponding wild types. Strains B s −2 , dar 2 − and dar 4 − were able to propagate certain ultraviolet-irradiated bacteriophages, whereas the other mutants hcr − , dar 1 − , dar 3 − , dar 5 − and dar 6 − were unable to do so. According to the results of the conjugation experiments each mutant appears to belong to one of three distinct linkage groups. The markers B s −2 , dar 3 − , dar 2 − and hcr − are located in a region between the markers thr and ile ; the markers dar 1 − and dar 6 − are closely linked to gal ; the markers dar 4 − and dar 5 − are closely linked to his . In addition the gene phr , that controls the recovery from lethal UV damage by subsequent illumination with visible light (photoreactivation), was found to be closely linked to gal .

1. 1. After treatment of a strain of Escherichia coli K 12 with the mutagenic agent N-methyl-N′-nitro-N-nitrosoguanidine, mutants were selected which divide normally at 30° but which show a strong inhibition of cell division during growth at 42°. 2. 2. According to their phenotype at elevated temperature the mutants were divided into the following classes. 1. I. Mutants that showed a complete inhibition of cell division and had no colony forming ability at 42°. 2. II. Mutants that showed an incomplete inhibition of cell division and formed colonies at 42° that consisted essentially of long filaments. 3. III. Mutants with no colony forming ability at 42° and in which the inhibition of cell division was accompanied and probably caused by an inhibition of nucleic acid synthesis. 1. 3. Mutants falling into the first class and not subject to inhibition of nucleic acid synthesis were studied in more detail. The temperature-induced inhibition of cell division, unlike the inhibition of cell division induced by ultraviolet irradiation, was not reversed by treatment with the agent pantoyl-lactone. Both inherited properties, sensitivity of temperature-induced and radiation-induced inhibition of cell division, are chromosomally determined; the corresponding genes however are located at different sites on the bacterial chromosome.

Bacteriophage Mu grown on Escherichia coli K12 (Mu. K) is restricted by wild type Citrobacter freundii. In two C. freundii mutants, where the restriction of foreign F′ factors is absent (de Graaff and Stouthamer, 1971), the restriction for Mu. K, although at a lower level, still exists. Consequently two host specificity systems exist in C. freundii, one affecting mainly the acceptance of foreign plasmidal and chromosomal DNA and one affecting foreign DNA of bacteriophage Mu. Mu is able to lysogenize C. freundii and to induce mutations at random in its chromosome. Furthermore Mu is able to promote the mobilization of the C. freundii chromosome in strains carrying F′ factors. Mu promoted integration of F ts 114 lac+ into the C. freundii chromosome was observed, resulting in the formation of stable Hfr strains. In this way it is possible to devise a method for chromosome transfer in other genera than E. coli to which plasmids of E. coli can be transferred, but in which no chromosome mobilization is possible because of poor DNA homology between the foreign plasmid and the host chromosome.

The uvrA6 mutation has been cloned on a multicopy plasmid by using a chloramphenicol resistance marker introduced next to the uvrA gene in the Escherichia coli chromosome. The mutation was shown to reside in the N-terminal part of the uvrA gene. Sequencing part of this region of the mutant gene revealed a frameshift mutation at positions 207 to 209, which leads to a stop codon at position 262. A marker rescue experiment showed that this frameshift is the only mutation responsible for the UV-sensitive phenotype of the UvrA6 mutant. The method presented is suitable for the cloning of every chromosomal uvrA mutation and can be useful for the study of the functional domains of the UvrA protein.

A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed. Its characteristics have been compared to a similar vector lacking the EBV sequences. (a) The EBV+ vector is maintained as an episome with a copy number of approx. 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line. (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique. (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell. (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell. (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed. (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system. (g) Rescue of the episomal vector from transfected cells can be readily achieved.

Bacteriophage lambdab2att2 [lambdab2cI857intam6(deltabioAB)bioFCD+uvrB+phr+] codes for a function(s) that confers UV resistance (Uvr+) and reactivation of irradiated phage (Hcr+) to an Uvr-Hcr-Escherichia coli strain. It was demonstrated that these functions are expressed under the control of bacterial regulatory elements located on lambdab2att2 DNA. The location of the E. coli uvrB gene on the DNA of this transducing phage was established by heteroduplex and restriction-enzyme analyses. Recombinant DNA molecules were constructed in vitro from plasmid pMB9 (Tcr), as the vector, and an EcoRI fragment (Eco-RI-F) of lambdab2att2 DNA. The resulting plasmid, designated pNP5, has a molecular weight of 5.1 X 10(6) and replicates in a relaxed fashion. Transformation of E. coli uvrB with plasmid pNP5 resulted in clones that are Uvr+ Tcr. Irradiation of bacteria transformed with plasmid pNP5 with low UV doses revealed a complete restoratation of the Uvr+ phenotype by the presence of the cloned EcoRI-F DNA, while only a partial restoration was observed after irradiation with high UV doses. Likewise, the Hcr+ character was also partially restored due to the presence of pNP5. No correlation was found between the acquired Uvr+, Hcr+ properties, and the presence of correndonuclease II activity in an extract of bacteria that harbor plasmid pNP5.

An Escherichia coli mutant has been isolated (Hek) in which the kil gene of bacteriophage Mu is not functionally expressed. The hek locus has been mapped between rpoD (66.2 min) and argR (69.5 min) on the E. coli chromosome. No influence of the hek mutation on phage or E. coli development could be detected.

The genome of bacteriophage Mu can integrate randomly in the chromosome of its host Escherichia coli, thereby causing mutations (Taylor, 1963). Random integration seems a more widespread phenomenon, since it was found that other DNA elements also show a more or less random integration: insertion sequences (IS) and transposons (Tn).

Genetic analysis of a mutant of Escherichia coli resistant to the antibiotic mocimycin is presented. This resistance is due to alterations in both tuf genes coding for the elongation factor Tu. Mocimycin resistance is recessive. Bacteria carryong only one tuf gene from the resistant mutant are still mocimycin sensitive. If the mutant gene is the tufA gene, the seisitive cells can be made resistant through inactivation of the tufB gene by insertion of the bacteriophage milliunits genome. Conditional mocimycin-resistant mutants ban also be isolated when the tufB gene is altered by an amber or a temperature-sensitive mutation. When only the tufB allele from the original mocimycin-resistant mutant is present, inactivation of the wild-type tufA gene fails to give viable mocimycin-resistant progeny. We conclude that the tufA mutant allele codes for a functional mocimycin-resistant EF-Tu, whereas the mutant tufB gene does not code for a functional product.

Mutation induction by cis-Pt(NH3)2Cl2 (cisplatin) has been shown to be absent in E.coli strains carrying a deletion of the uvrB gene (1). This suggested that excision-repair, which is normally thought to be error-free, is involved in mutation induction with cisplatin. Here, the role of the excision repair enzymes UvrA, UvrB and UvrC is investigated using E.coli strains with different repair capacities. It is shown that cisplatin induced mutagenesis is dependent both on UvrA and UvrB but not on UvrC. Of the UvrB enzyme the N-terminal 113 aminoacids are sufficient for mutation induction by cisplatin.

Mutants of Pseudomonas aeruginosa have been isolated which have a reduced ability to be lysogenized by bacteriophages. These mutants also have a strongly reduced ability to form recombinants when mated with a suitable donor strain and for this reason are indicated as Rec−. One of the Rec− strains, named R 57−58 which was studied in more detail, is only lysogenized when infected with high multiplicities of bacteriophages; cells which have been lysogenized cannot be induced by ultraviolet light. It has been found that strain R 57–58 is unable to grow on complete medium at 42° and that the inability to form colonies at this temperature is caused by a mutation that prevents DNA synthesis. Loss of the ability to recombine and thermo-sensitivity appear to related because temperature markedly influences the recombination properties of the strain. Moreover genetic data show that the rec mutation and the mutation which leads to thermosensitivity are identical or very closely linked.

• Various properties of four mutants of Escherichia coli CR34, defective in genetic recombination (Rec−) were studied. • The mutants are more sensitive to UV- and X-irradiation, in comparison with the wild-type strain, but are able to propagate irradiated bacteriophage λ like the wild type. • The mutants are able to conjugate with F′ strains and to maintain the F factor. In crosses with Hfr strains the appearance of stable recombinants is delayed until the entrance of the allelic rec+ marker of the donor in the Rec− recipient. Therefore the rec+ allele must be dominant over the rec− allele. This allows the determination of the approximate position of the rec− mutations on the chromosome. Three different sites appear to be involved; two mutations (rec35−, rec38−) are most probably located near thr, one (rec36−) between Sr and his and one (rec34−) probably between try and his. • Derivatives of the Rec− mutants, lysogenic for λ, show different UV prophage induction patterns. • Recombination among genetically-marked λ phages is not restricted in any of the Rec− strains.