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Knowledge exchange is a broad and consequential undertaking, analysed by diverse scholars, and rapidly growing as a field of academic practice. Its remit is to strengthen ties between research generators and users to support better material outcomes for society. This review paper considers how this increasingly codified academic field might engage with the research-practice concerns identified in the Indigenous and decolonial literature. We do so by bringing the two literature sets together for analysis, noting they are not mutually exclusive. We reveal how addressing discrimination towards Indigenous peoples from withinthe knowledge exchange field requires a fundamental reconsideration of the biases that run throughthe field’s structures and processes. We prioritise two connected framing assumptions for shifting—jurisdictional and epistemological. The first shift requires a repositioning of Indigenous peoples as political–legal entities with societies, territories, laws and customs. The second shift requires engagement with Indigenous expert knowledge seriously on its own terms, including through greater understanding about expert knowledge creation with nature. These shifts require taking reflexivity much further than grasped possible or appropriate by most of the knowledge exchange literature. To assist, we offer heuristic devices, including illustrative examples, summary figures, and different questions from which to start the practice of knowledge exchange. Our focus is environmental research practice in western Anglophone settler-colonial and imperial contexts, with which we are most familiar, and where there is substantial knowledge exchange literature.

Evaluating impact of animal agriculture on air quality has been the focus of recent research. Ammonia (NH3) volatilization occurs when undigested protein in feces and urea in urine is broken down by bacteria and enzymes. Information regarding NH3emission from equine facilities is limited, and effects of CP intake on NH3emissions have not been investigated. Nine mature geldings were used in a 3 × 3 replicated Latin square design study to determine effects of dietary CP on potential NH3losses from feces and urine. We hypothesized feeding horses above the CP requirement would result in an increase in NH3emissions from urine and feces and different bedding materials would affect NH3emissions from urine. Diets were formulated using different ratios of bahiagrass (Paspalum notatum) and Tifton-85 bermudagrass (Cynodon dactylon) hays, and a commercial vitamin mineral supplement to provide 3 different CP concentrations and labeled in relation to each other: LOW-CP, MED-CP, and HIGH-CP (10.6%, 11.5%, and 12%, respectively). Each study period consisted of an 11-d diet adaptation phase, followed by a 3-d total collection of urine and feces. To determine total nitrogen (TN) and urea-N concentrations, samples were pooled by period (n= 9). For in vitro determination of NH3concentrations, urine and fecal samples were pooled within period by diet (n= 3) and mixed with either wheat straw or wood shavings. Ammonia emission of these samples was measured using a vessel system with an airflow rate (2.5 L/min) at 20°C over a 7-d period. Concentration of NH3in each vessel was measured using a photoacoustic multigas analyzer. Temperature, airflow rate, and NH3concentration in each vessel were used to calculate NH3emission rate (ER). Data were analyzed using a mixed model ANOVA with repeated measures. Urinary TN and urea-N excretion increased as CP intake increased (P< 0.0001). Vessel urinary NH3concentrations were not different across diets (P= 0.1225), ranging from 55.48 ppm (LOW-CP) to 101.14 ppm (HIGH-CP); however, they differed between bedding types (P< 0.0001), with straw higher than shavings (97 vs. 73.5 ppm, respectively). Cumulative urinary NH3ER tended to be different across diets (P= 0.0550) ranging from 5.87 g/m2to 9.97 g/m2and bedding types (P= 0.0129), with straw being higher than shavings (11.1 vs. 6.9 g/m2, respectively). Overfeeding CP to horses can lead to increased urinary TN and urea-N excretion, which could lead to greater of NH3in the atmosphere.

The MW7.1 Darfield (Canterbury) earthquake, 4 September 2010, generated widespread hydrological effects in New Zealand ranging from instantaneous changes of piezometric levels, to more sustained postseismic changes in spring flow, river discharge and groundwater levels, and increased turbidity and declined yields of water abstracted from wells. Four years later, piezometric levels remained elevated in deeper (>40 m) aquifers along the north-western (upper) side of the Canterbury Plains near the Greendale Fault, with changes in mean piezometric level reaching +13 m. Linear reservoir modeling (eigen modeling) suggests that sustained high groundwater was not the result of changes in abstraction or land surface recharge. Step-drawdown tests at six wells within 15 km of Greendale Fault were carried out prior to the earthquake and were retested following fault rupture. Eden-Hazel analysis of discharge/drawdown relationships discriminates potential sources of head losses, and how these changed (or otherwise) as a result of the earthquake. Objective application of Eden-Hazel analysis provided confidence levels for the interpretation, including when step tests provide reliable/unreliable estimates of transmissivity change. Increases in both aquifer losses and well losses were observed in four wells, reflecting both a change in sediment transmissivity and decrease in well efficiency. At two locations, the data were unable to provide results that can be interpreted with confidence. As the majority of local groundwater flow occurs through high-permeability open framework gravel lenses, we suggest that reduction in the permeability of these gravels, due to fine-sediment incursion, is the cause of the reduction in transmissivity and increase in well losses. Piezometric levels showed sustained changes as a result of a Mw7.1 earthquakeStep testing confirmed a reduction in transmissivity in the region of sustained groundwater riseIt is proposed that pore space in high-permeability open framework gravels is reduced by fine-sediment incursion

Aims:: The association between central obesity and insulin resistance reflects the properties of visceral adipose tissue. Our aim was to gain further insight into this association by analysing the lipid composition of subcutaneous and omental adipose tissue in obese women with and without insulin resistance. Methods:: Subcutaneous and omental adipose tissue and serum were obtained from 29 obese non-diabetic women, 13 of whom were hyperinsulinemic. Histology, lipid and gene profiling were performed. Results:: In omental adipose tissue of obese, insulin-resistant women, adipocyte hypertrophy and macrophage infiltration were accompanied by an increase in GM3ganglioside and its synthesis enzyme ST3GAL5; in addition, phosphatidylethanolamine (PE) lipids were increased and their degradation enzyme, phosphatidylethanolamine methyl transferase (PEMT), decreased. ST3GAL5was expressed predominantly in adipose stromovascular cells and PEMTin adipocytes. Insulin resistance was also associated with an increase in PE lipids in serum. Interpretation:: The relevance of these findings to insulin resistance in humans is supported by published mouse studies, in which adipocyte GM3ganglioside, increased by the inflammatory cytokine tumour necrosis factor-a, impaired insulin action and PEMT was required for adipocyte lipid storage. Thus in visceral adipose tissue of obese humans, an increase in GM3ganglioside secondary to inflammation may contribute to insulin resistance and a decrease in PEMT may be a compensatory response to adipocyte hypertrophy.

AbstractThe Army Behavioral Health Integrated Data Environment (ABHIDE) is a suicide registry containing extensive data on U.S. Army Soldiers with suicidal behavior. Development of the ABHIDE began in 2009 in response to the doubling of the Army suicide rate from 2004 to 2008. It now consists of 28 administrative data sets from 11 organizations throughout the Departments of the Army and Defense. Data include personnel variables, military characteristics, medical claims, criminal histories, characteristics of the suicidal events, and stressors. Through the ABHIDE, the U.S. Army is better able to answer requests for information, inform public health practice, and develop suicide prevention programs.

How, we might ask, are sport and religion connected? The truth is that, in Britain at least, these two cultural practices share a good deal of common ground primarily as a consequence of the role of Protestantism in the development of sport in Victorian times. Recent years have witnessed an increasing amount of academic discussion and debate around the sport–religion relationship. What this article aims to do is to map out the key historical features of this relationship while providing insight into the ways in which the Protestant Church continues to pursue sport as a form of outreach and ministry.

The authors investigated whether thoracic lymphatic pumping (TLP) after FluShield vaccination enhanced the production of anti-influenza immunoglobulins in elderly individuals, who are at particular risk for influenza. Osteopathic students and non-TLP-treated elderly subjects served as controls. Serum antibody titers were quantified with enzyme-linked immunosorbent assay, and hemagglutination inhibition assay, both of which generated comparable results. While approximately 70% of the younger controls had increased anti-influenza immunoglobulin production on vaccination, only 30% to 35% of the aged population had increased antibody production. There was no significant enhancement in anti-influenza immunoglobulin production in the TLP-treated subjects. The authors' findings suggest that TLP in conjunction with influenza vaccination does not enhance immunization against influenza in otherwise healthy and active populations. However, such techniques may be of value when applied in conjunction with vaccination to nonambulatory patients or on actual influenza exposure of at-risk individuals.

AIM: Inflammatory abdominal aortic aneurysms (IAAA) are a variant of abdominal aortic aneurysms, which are associated with an increased morbidity. The diagnosis of IAAA has traditionally been established with a combination of clinical and laboratory findings together with contrast enhanced CT. There is a high incidence of renal impairment in this group, and therefore contrast enhanced CT may be harmful.

Summary Cytotoxic chemotherapeutic agents, particularly the anthracyclines, are known to be cardiotixic, but toxic effects on the aorta have not previously been documented. In this study, diameters of ascending and descending thoracic aortae were measured by computerized tomography in 69 patients with lymphoma, before and after first-line treatment with one of 7 different regimes. Minor increases in aortic diameter over the study period due to the aging process were expected. These increases were greater than anticipated in both the ascending and the descending aortae after chemotherapy with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) and CVP (cyclophosphamide, vincristine, and prednisolone) regimes. Smaller changes, or changes which were not statistically significant, were noted after MVPP (mustine, vinblastine, procarbazine, and prednisolone), ChlVPP (chlorambucil, vinblastine, procarbazine, and prednisolone), ChlVP (chlorambucil, vincristine and prednisolone), mediastinal radiotherapy, and radiotherapy plus MVPP (MVPP/XRT). Cardiovascular damage associated with certain forms of cytotoxic therapy is not confined to the heart, but also affects the aorta.

The relationship between the cross-sectional area (CSA) and maximum voluntary isometric strength of the knee extensor muscles was measured in a group of untrained subjects (control group, n=30), a group of successful marathon runners (n=6) and a group of successful sprinters (n=6). All subjects were male, aged between 20 and 41 years. In the control group muscle strength was found to be positively correlated with lean body mass (p<0.01) and with muscle CSA as determined by computed tomography scanning of the leg at mid-thigh level (p<0.001). However, in spite of these significant relationships, there was considerable variability between subjects. The sprinters were significantly stronger than the endurance runners (p<0.01), but neither of the athletic groups differed significantly from the control group. Muscle CSA was greater in the sprinters than in the marathon runners but this difference was not significant. The ratio of muscle strength to CSA in the control group varied from 7.07 to 13.57. All the trained subjects fell within this normal range, but the sprinters' muscles were stronger per unit CSA than the muscles of the marathon runners (p<0.05). Since it is well established that the leg muscles of sprinters contain a high proportion of fast twitch (FT) muscle fibres whereas endurance runners have a high proportion of slow twitch (ST) fibres, these results suggest that the strength which can be produced by a muscle may depend on its fibre composition. Variation in the distribution of the different fibre types in the untrained population may therefore account at least in part for the wide variability observed in this group.

The maximum voluntary force (strength) which could be produced by the knee‐extensor muscles, with the knee held at a right angle, was measured in a group of healthy young subjects comprising twenty‐five males and twenty‐five females. Both legs were tested: data from the stronger leg only for each subject were used in the present study. Computed tomography was used to obtain a cross‐sectional image of the subjects' legs at mid‐thigh level, measured as the mid‐point between the greater trochanter and upper border of the patella. The cross‐sectional area of the knee‐extensor muscles was determined from the image obtained by computer‐based planimetry. The subjects' height and weight were measured. An estimate of body fat content was obtained from measurements of skinfold thicknesses and used to calculate lean body mass. Male subjects were taller (P less than 0.001), heavier (P less than 0.001), leaner (P less than 0.001) and stronger (P less than 0.001) than the female subjects. No significant correlation was found to exist between strength of the knee‐extensor muscles and body weight in the male or in the female subjects. In the male subjects, but not in the female group, there was a positive correlation (r = 0.50; P less than 0.01) between strength and lean body mass. Muscle cross‐sectional area of the male subjects was greater than that of the female subjects (P less than 0.001). The ratio of strength to cross‐sectional area for the male was 9.49 +/‐ 1.34 (mean +/‐ S.D.). This is greater but not significantly so, than that for females (8.92 +/‐ 1.11). In both male and female groups, there was a significant (P less than 0.01) positive correlation between muscle strength and cross‐sectional area. A wide variation in the ratio of strength to muscle cross‐sectional area was observed. This variability may be a result of anatomical differences between subjects or may result from differences in the proportions of different fibre types in the muscles. The variation between subjects is such that strength is not a useful predictive index of muscle cross‐sectional area.

The early detection of dementia carries implications for clinical management for patients and their families and is of utmost concern if an effective pharmacological treatment is to be found. The utility of an enhanced cued recall paradigm for predicting dementia in a group of elderly subjects was examined. Forty-five subjects referred for clinical evaluation who did not meet the criteria for dementia at initial assessment were reassessed 12-18 months later. Eighteen of these subjects were diagnosed as having possible or probable Alzheimer Disease at reassessment and the diagnostic status of 27 remained unchanged. At initial assessment the 'change' group performed more poorly than the 'no change' group on measures of retrieval, acquisition and retention derived from the cued recall procedure. As would be expected, the performance of the 'no change' group remained stable over time whereas the performance of the 'change' group deteriorated, resembling the performance of a group of subjects with mild possible or probable Alzheimer disease. When the sensitivity and specificity of the memory variables were examined, the retrieval measure (i.e., free recall) appeared most useful as an early predictor of dementia. Continued longitudinal evaluations of subjects with questionable dementia are needed to address more fully the natural history of early memory changes associated with dementia.

One hundred ambulant outpatients with active, endoscopically proven peptic ulceration entered a double-blind trial of either tripotassium-dicitratobismuthate or placebo. Thirty-four patients had gastric ulceration, 56 had duodenal ulceration, three had both gastric and duodenal ulcers, and two had stomal ulceration. Five patients with gastric ulceration were withdrawn from the trial. Three patients with both gastric and duodenal ulceration and two patients with stomal ulceration were excluded from statistical analysis. After 28 days of tripotassium-dicitratobismuthate 94% of gastric ulcer patients had significant endoscopic healing (P<0.01). Although 75% of duodenal ulcers healed after 28 days of tripotassium-dicitratobismuthate, this was not statistically significant because of a 60% rate of healing with placebo. Tripotassium-dicitratobismuthate produced a significantly quicker symptomatic response in duodenal ulcer patients (P<0.01). No serious side effects were recorded, and patient acceptability was high. It is concluded that tripotassium-dicitratobismuthate is an effective agent for promoting gastric ulcer healing and for symptomatic relief in duodenal ulceration.

Exposure of BALB/c mice to mosquitoes infected with irradiated Plasmodium berghei confers protective immunity against subsequent sporozoite challenge. Immunized mice challenged with viable sporozoites develop parasitemia when treated orally with substrate inhibitors of nitric oxide synthase (NOS). This suggests that the production of nitric oxide (NO) prevents the development of exoerythrocytic stages of malaria in liver. Liver tissue from immunized mice expressed maximal levels of mRNA for inducible NOS (iNOS) between 12 and 24 h after challenge with sporozoites. Intraperitoneal injection of neutralizing monoclonal antibody against interferon gamma (IFN-gamma) or in vivo depletion of CD8+ T cells, but not CD4+ T cells, at the time of challenge blocked expression of iNOS mRNA and ablated protection in immunized mice. These results show that both CD8+ T cells and IFN-gamma are important components in the regulation of iNOS in liver which contributes to the protective response of mice immunized with irradiated malaria sporozoites. IFN-gamma, likely provided by malaria-specific CD8+ T cells, induces liver cells, hepatocytes and/or Kupffer cells, to produce NO for the destruction of infected hepatocytes or the parasite within these cells.

The Early Devonian Walhalla Group is a turbidite sequence deposited on the eastern margin of the Melbourne Trough. Five lithofacies were identified: (1) massive sandstones; (2) turbidites with a basal Ta division; (3) turbidites with a basal laminated division (Tb or Td); (4) fine grained, rippled sandstones interbedded with shales; and (5) shales and siltstones. Episodic reworking by water currents has produced reworking features such as wave-current and unidirectional current ripples, sharp topped turbidites and interrupted Bouma sequences. The reworking was attributed to storm currents indicating that the sequence was deposited above storm-weather wave-base. Palaeobathymetric calculations, based on wave ripple geometry, are consistent with geological observations, and suggest that deposition took place in 65-90 m of water. The relatively shallow water depths implied, combined with the general areal continuity of lithofacies, suggested that the turbidites are sheet-like deposits originating from a distribution of line sources. It was therefore suggested that the sequence outcropping in the vicinity of Walhalla was deposited in either a wave-cut shelf or shallow marine fan environment.

1. Computed tomography has been used to establish the proportions of fat, muscle and bone present in the normal human forearm. 2. Subjects were healthy young volunteers, eight males and eight females. A series of six cross-sectional scans at right angles to the long axis of the forearm was obtained, the scans being made at equidistant intervals between the olecranon process and the ulnar styloid. 3. The volumes occupied by fat, muscle and bone were calculated for the complete forearm. Total forearm volume calculated by this method agreed closely with that measured by water displacement. 4. For the male subjects, forearm composition was 72.1 ± 4.4% muscle (mean ± SD), 15.0 ± 5.3% fat and 12.9 ± 1.2% bone. Female forearms had less muscle (P<0.001) and more fat (P<0.001) than those of the male subjects: 58.5 ± 4.0% muscle, 29.3 ± 5.0% fat and 12.3 ± 1.4% bone. 5. Forearm muscle volume was significantly correlated (r= 0.75; P<0.05) with lean body mass in the male subjects; the range of values for lean body mass in the female subjects was too small to permit calculation of the equivalent relationship. 6. Forearm fat content, as a percentage of total volume, was proportional to whole body fat content as estimated from skinfold thickness (males, r = 0.84; P< 0.01; females, r = 0.77; P< 0.05).

The considerable investment which has been put into research on the effect of light on plant growth is at last showing a substantial return for the greenhouse industry. The British grower, faced with increasing economic pressures over the past few years, has grasped the opportunity of using this accumulated knowledge and, with the help of various interested research bodies and lamp manufacturers, has created a new lighting technology for plant production. The various lighting techniques at present used in commercial horticulture are discussed together with their economic implications for the grower.

Tak Tent is a cancer support organisation consisting of 14 groups of which 11 are based in Scotland. In 1985, a survey was conducted among those attending the Scottish groups. 146 (79%) of the groups' members completed survey questionnaires. The results showed that Tak Tent's membership mainly comprised cancer patients (36%), relatives of patients (34%) and professionals involved in cancer care (21%). Women outnumbered men 3 to 1 and most of the membership belonged to social classes I, II or III. The groups appeared to be meeting their members' expectations of them to varying degrees. Respondents were satisfied that group membership had allowed them to make new friends, find out more about cancer and meet others facing similar difficulties. They were less certain that participation in a group had enabled them to learn how to cope better with cancer, share their problems with others or provide support for others to the extent they had anticipated.

Student motivation and knowledge restructuring are examined in the light of experience with engineering design projects. The work is related to existing experience with a broad range of observations resulting from educational experiments. The results of student responses to project work clearly identify concepts which are avoided by students when solving engineering problems. It is conjectured that these orphan concepts provide signposts to a better learning environment. The students' responses also suggest that working in a familiar environment, where an informal knowledge base already exists, is a strong positive motivator for knowledge restructuring.

Human monocytes and macrophages are nondividing cells that serve as a major reservoir for human immunodeficiency virus (HIV) at all stages of infection. To investigate viral-mediated gene delivery as a means of inhibiting HIV replication in human monocytes, a replication-incompetent herpes simplex virus vector was developed that expressed human interferon alpha. Monocytes infected with this herpes simplex virus vector and then challenged with HIV showed dramatically reduced cytopathic effects and HIV replication compared to control treated monocytes. Similar effects on HIV replication were observed if monocytes were first infected with HIV and then treated with the recombinant vectors. These results demonstrate that replication-incompetent herpes simplex virus gene delivery of interferon alpha directly to human monocytes can greatly decrease HIV replication and suggest that such a vector might deliver therapeutically important genes directly to sites of HIV infection.

The DNA sequence of a vaccinia virus late gene contains an open reading frame that corresponds to the 28,000-dalton (28K) polypeptide made by in vitro translation of hybrid-selected mRNA. To further characterize the protein product of this late gene, we cloned a segment of DNA containing part of the open reading frame into a bacterial expression vector. The fusion protein produced from this vector, containing 151 amino acids of the predicted vaccinia virus protein, was used to immunize rabbits. The resulting antiserum specifically bound to a major 25K structural protein that is localized in the core of vaccinia virions, as well as to a 28K protein found in infected cells. Pulse-chase experiments indicated that the 25K core protein is originally made as a 28K precursor.

A subset of vaccinia virus genes are expressed only after DNA replication. To investigate the regulation of such transcriptional units, a representative gene encoding a major late polypeptide (Mr, 28,000) was mapped and sequenced. Translatable mRNAs were heterogeneous in length and overlapped several early genes downstream. The 5' end of the message was located, and the DNA segment upstream was excised and ligated to the coding sequence of the easily assayable procaryotic chloramphenicol acetyltransferase gene. The resulting chimeric gene was recombined into the thymidine kinase locus of the vaccinia virus genome, and infectious recombinant virus was isolated. Both the time of chloramphenicol acetyltransferase synthesis in infected cells and the requirement for DNA replication indicate that the sequence upstream of the late gene contains cis-acting transcriptional regulatory signals.

This study has examined muscle strength and cross-sectional area in a group of 35 healthy untrained male subjects and 8 subjects who had been engaged in a strenuous weight-training programme. The maximum voluntary knee extension force which could be produced by the untrained subjects was 742 +/- 100 N (mean +/- SD). The trained subjects could produce a significantly (p less than 0.001) greater force (992 +/- 162 N). Cross-sectional area of the knee-extensor muscle group was 81.6 +/- 11.8 cm2 in the untrained subjects and 104.1 +/- 12.3 cm2 in the trained subjects (p less than 0.001). In the untrained subjects, a significant correlation existed between strength and muscle cross-sectional area (r = 0.56, p less than 0.001). In the same group of subjects, there was a significant inverse relationship between muscle cross-sectional area and the ratio of strength to cross-sectional area (r = 0.55, p less than 0.001). The mean ratio of strength to cross-sectional area was 9.20 +/- 1.29 for the untrained group whereas for the trained group this ratio was 9.53 +/- 1.01. It is suggested that the inverse relationship between strength per unit cross-sectional area and cross-sectional area results in part from an increased angle of pennation in the larger muscles.

To investigate the cis-acting sequence elements that are involved in the regulation of herpes simplex virus type 1 late gene expression, linker-scanning mutations were constructed in the promoters of the glycoprotein C and glycoprotein H genes. Each promoter mutation was inserted upstream of the Escherichia coli lacZ gene in a recombinant virus, and the relative activities of beta-galactosidase expressed from individual recombinant viruses were compared. This analysis identified three sequence elements in each promoter: a TATA element, an element that overlapped the start of transcription, and an element downstream from the start of transcription. Primer extension analysis confirmed these results and showed that mutations in either the TATA element or the initiation sequence could eliminate normal transcription initiation. Analysis of expression from hybrid promoters revealed that the TATA and the initiation elements were interchangeable, at least when correctly aligned, and that the initiation element plays a pivotal role in determining the actual site of transcription initiation.

To investigate the cis-acting sequence elements that are involved in the regulation of herpes simplex virus type 1 late-gene expression, recombinant viruses were constructed that express the Escherichia coli lacZ gene from the promoter of the glycoprotein H (gH) gene. Deletion experiments established an upstream boundary for the gH promoter of no more than 83 bp from the start of gH transcription and showed that the promoter sequences did not overlap with coding sequences of the upstream thymidine kinase (tk) gene. Sequences of the tk gene previously shown to be required for efficient processing of the tk transcript were essential for expression form the gH promoter and included a TATA-like element. In addition, the gH TATA element was specifically mutagenized to substitute the TATA elements of immediate-early, early, and other late viral promoters for the gH TATA element. The results indicated that the TATA element was an interchangeable component of herpes simplex virus type 1 promoters and did not regulate temporal expression.

The role of the 5' noncoding region of the herpes simplex virus type 1 glycoprotein C (gC) gene in viral gene expression was investigated with recombinant herpesviruses that contained the bacterial beta-galactosidase gene under the control of the gC promoter-regulatory region. Each of these viruses had the same DNA sequences from the start of gC transcription upstream to -114 but had variable segments of the downstream 140-base-pair sequence that is between the start of gC transcription and translation. Analysis of beta-galactosidase expression and mRNA synthesis from these viruses demonstrated the importance of DNA sequences from the start of gC transcription downstream to +38 for optimal expression from the gC promoter.

A putative promoter region, extending from 218 base pairs (bp) before (-218) to 10 bp after (+10) the RNA start site of a vaccinia virus gene encoding an Mr-28,000 precursor of a core polypeptide that is expressed only after the onset of DNA replication was linked to DNA coding for the procaryotic enzyme chloramphenicol acetyltransferase (CAT). When this chimeric gene was inserted into the genome of vaccinia virus, the infectious recombinant expressed CAT in a regulated fashion. A series of deletions starting upstream of the promoter region and extending toward the RNA start site were made. The effects of these mutations on CAT expression were examined in cells infected with recombinant viruses and confirmed in a helper virus-dependent transient assay system. A gradual reduction in CAT expression occurred as the deletions extended from -61 to -18. Mutants that retained 18 bp before and 10 bp after the RNA start site still expressed CAT as a late gene product, although at a submaximal level. A further 5'-to-3' deletion of 10 bp reduced CAT expression to background levels. To demonstrate that the effect on expression was not simply due to the bringing of upstream inhibitory sequences closer to the RNA start site, a point mutation substituting a G for the A at -12 was made. The sharp decrease in CAT expression indicated the importance of a run of eight A residues located between -15 and -7. Evidence that these mutations affected the level but not the site of transcriptional initiation was demonstrated by analysis of the 5' ends of the mRNAs from infected cells. The short DNA sequence required for accurate and temporally regulated transcription suggests that the same or overlapping signals are used for both aspects of this process.

The sequence of the 8,600-base-pair HindIII H fragment, located at the center of the vaccinia virus genome, was determined to analyze several late genes. Seven major complete open reading frames (ORFs) and two that started from or continued into adjacent DNA segments were identified. ORFs were closely spaced and present on both DNA strands. Some adjacent ORFs had oppositely oriented overlapping termination codons or contiguous stop and start codons. Nucleotide compositional analysis indicated that the A-T frequency was consistently lowest in the first codon position. The sizes of the polypeptides predicted from the DNA sequence were compared with those determined by polyacrylamide gel electrophoresis of cell-free translation products of mRNAs selected by hybridization to cloned single-stranded DNA segments or synthesized in vitro by bacteriophage T7 RNA polymerase. Six transcripts that initiated within the HindIII H DNA fragment were detected, and of these, four were synthesized only at late times, one was synthesized only early, and one was synthesized early and late. The sites on the genome corresponding to the 5' ends of the transcripts were located by high-resolution nuclease S1 analysis. For late genes, the transcriptional and translational initiation sites mapped within a few nucleotides of each other, and in each case the sequence TAAATGG occurred at the start of the ORF. The extremely short leader and the absence of A or G in the -3 position, relative to the first nucleotide of the initiation codon, distinguishes the majority of vaccinia virus late genes from eucaryotic and vaccinia virus early genes.

Nucleotide sequencing of a 1,300-base-pair vaccinia virus DNA segment previously shown to contain a thymidine kinase (TK) gene revealed an uninterrupted reading frame of 177 codons capable of producing a polypeptide with a molecular weight of 20,102. Mapping of the TK mRNA by primer extension indicated a unique 5' end that precedes the initiation codon by only six nucleotides. Multiple 3' ends within a 10-nucleotide region, about 30 nucleotides beyond the termination codon, were located by nuclease digestion of DNA-RNA hybrids, and the length of the TK transcript, exclusive of polyadenylate, was estimated to be approximately 570 nucleotides. The region preceding the TK mRNA start site is extremely A + T rich and has sequence homologies with three other early genes. Genetic information is so compressed in this region of the DNA that the putative transcriptional regulatory sequence of the TK gene overlaps the coding sequence of a late gene. Only nine nucleotides separate the termination codon of the late gene from the initiation codon of the TK gene. Downstream, 66 nucleotides separate the TK termination codon from the apparent initiation codon of another early gene. The nature of three independent TK- mutants was revealed by nucleotide sequencing. Each has a nucleotide reiteration leading to a +1 frameshift and a nonsense codon downstream. The location of one frameshift mutation provided evidence that the first ATG is used for initiation of protein synthesis.

Enzymatically active thymidine kinase (TK) was made in reticulocyte lysates programmed with early vaccinia mRNA that hybridized to plasmid recombinants containing either of two adjacent small DNA subsegments of the viral HindIII-J fragment. The map position of an early polypeptide, with a molecular weight of 19,000 (19K), coincided precisely with that of the TK. The absence of the 19K polypeptide in cell-free translation products of hybridization-selected mRNAs from several TK-negative mutants provided an independent identification of the TK polypeptide. The small size of the TK polypeptide of vaccinia virus distinguishes it from that of procaryotes, eucaryotes, and herpesvirus. Five early mRNAs of 3,840, 2,390, 1,790, 1,070, and 590 nucleotides were mapped within the HindIII-J fragment by RNA blotting and nuclease S1 digestion of RNA-DNA hybrids. The RNAs of 590 and 2,380 nucleotides were found to have 5' coterminal ends and represent major and minor forms, respectively, of the TK message. The 3' end of the minor TK mRNA appeared to be coterminal with the 3' end of the 1,790-nucleotide transcript which encodes a 41K polypeptide. The 1,070-nucleotide RNA was identified as the message for a 21K polypeptide. All of these RNAs, including the two forms of the TK message, were made by the putative TK-negative nonsense mutants.

The Bacillus spo0H gene codes for sigma H, which, as part of the RNA polymerase holoenzyme E sigma H, is responsible for the transcription of several genes which are expressed at the beginning of the sporulation process. In this communication, we examined the regulation of the spo0H gene of Bacillus subtilis by using lacZ reporter gene assays, quantitative RNA determinations, and Western immunoassay. The expression of the spo0H gene increases as the culture enters the mid-logarithmic stage of growth. This increased expression requires the genes spo0A, spo0B, spo0E, and spo0F, and the requirement for at least spo0A and spo0B can be bypassed when the abrB gene is mutated. The expression of the spo0H gene is constitutive in the presence of the abrB mutation, being expressed at higher levels during vegetative growth. In addition, the sof-1 mutation, in the spo0A structural gene, can bypass the need for spo0F in spo0H expression. The transcriptional start site of spo0H was determined by using RNA made in vivo as well as in vitro. These studies indicate that spo0H is transcribed by the major vegetative RNA polymerase, E sigma A. spo0H RNA and sigma H levels during growth are not identical to each other or to the pattern of expression of spoVG, a gene transcribed by E sigma H. This suggests that spo0H is regulated posttranscriptionally and also that factors in addition to sigma H levels are involved in the expression of genes of the E sigma H regulon.