A neurogenetic model for the study of schizophrenia spectrum disorders: the International 22q11.2 Deletion Syndrome Brain Behavior Consortium.

Rare copy number variants contribute significantly to the risk for schizophrenia, with the 22q11.2 locus consistently implicated. Individuals with the 22q11.2 deletion syndrome (22q11DS) have an estimated 25-fold increased risk for schizophrenia spectrum disorders, compared to individuals in the general population. The International 22q11DS Brain Behavior Consortium is examining this highly informative neurogenetic syndrome phenotypically and genomically. Here we detail the procedures of the effort to characterize the neuropsychiatric and neurobehavioral phenotypes associated with 22q11DS, focusing on schizophrenia and subthreshold expression of psychosis. The genomic approach includes a combination of whole-genome sequencing and genome-wide microarray technologies, allowing the investigation of all possible DNA variation and gene pathways influencing the schizophrenia-relevant phenotypic expression. A phenotypically rich data set provides a psychiatrically well-characterized sample of unprecedented size (n=1616) that informs the neurobehavioral developmental course of 22q11DS. This combined set of phenotypic and genomic data will enable hypothesis testing to elucidate the mechanisms underlying the pathogenesis of schizophrenia spectrum disorders.

Is obesity still increasing among pregnant women? Prepregnancy obesity trends in 20 states, 2003-2009.

OBJECTIVE: To estimate trends in prepregnancy obesity prevalence among women who delivered live births in the US during 2003-2009, by state, age, and race-ethnicity. METHODS: We used Pregnancy Risk Assessment Monitoring System (PRAMS) data from 2003, 2006, and 2009 to measure prepregnancy obesity (body mass index [BMI]≥30kg/m(2)) trends in 20 states. Trend analysis included 90,774 records from 20 US states with data for all 3 study years. We used a chi-square test for trend to determine the significance of actual and standardized trends, standardized to the age and race-ethnicity distribution of the 2003 sample. RESULTS: Prepregnancy obesity prevalence increased by an average of 0.5 percentage points per year, from 17.6% in 2003 to 20.5% in 2009 (P<0.001). Obesity increased among women aged 20-24 (P<0.001), 30-34 (P=0.001) and 35 years or older (P=0.003), and among non-Hispanic white (P<.001), non-Hispanic black (P=0.02), Hispanic (P=0.01), and other women (P=0.03). CONCLUSION: Overall, prepregnancy obesity prevalence continues to increase and varies by race-ethnicity and maternal age. These findings highlight the need to address obesity as a key component of preconception care, particularly among high-risk groups.

Real-world disparities and ethical considerations with access to CFTR modulator drugs: Mind the gap!

The third Sustainable Development Goal (SDG), to ensure healthy lives and promote well-being for all at all ages, has particular relevance and implementation challenges amongst people living with rare diseases such as cystic fibrosis (CF). Although the treatment and projected outcome of CF has significantly improved with the advent of CF transmembrane conductance regulator protein modulator (CFTRm) therapy, there remains significant global inequality with regards to access to these life-saving and life-altering drugs. Elexacaftor, tezacaftor, and ivacaftor (ETI) triple combination therapy, first licensed in the United States in 2019, has rapidly become the standard of care for children aged 6 years and older in most high-income countries for individuals with CFTR variants responsive to ETI. Negotiated agreements for access to ETI are currently in place in North America,Europe, Israel ,Australia and New Zealand. However, less priority has been given to negotiate agreements for access to CFTRm in low-middle income countries(LMIC) with significant CF populations such as Central and South America, India, the Middle East, and Southern Africa. These countries and individuals living with CF are therefore effectively being left behind, in direct conflict with the stated principle of the 2030 SDGs. In this review, we highlight the current global inequity in access to CFTRm drugs and its impact on widening disparities between high-income countries and LMIC in CF outcomes and survival. We further discuss the reasons for this inequity and explore the ethical- and human rights-based principles and dilemmas that clinicians, families, governments, and healthcare funders must consider when prioritizing fair and affordable access to expensive CFTRm drugs. Lastly, we propose possible solutions to overcoming the barriers to accessing affordable CFTRm drugs in LMIC and illustrate with examples how access to drug therapies for other conditions have been successfully negotiated in LMIC through innovative partnerships between governments and pharmaceutical industries.

Risk factors and outcomes of extubation failure in a South African tertiary paediatric intensive care unit.

Background: Extubation failure contributes to poor outcome of mechanically ventilated children, yet the prevalence and risk factors have been poorly studied in South African (SA) children. Objectives: To determine the prevalence, risk factors and outcomes of extubation failure in an SA paediatric intensive care unit (PICU). Methods: This was a prospective, observational study of all mechanically ventilated children admitted to a tertiary PICU in Cape Town, SA. Extubation failure was defined as requiring re-intubation within 48 hours of planned extubation. Results: There were 219 episodes of mechanical ventilation in 204 children (median (interquartile range (IQR)) age 8 (1.6 - 44.4) months). Twenty-one of 184 (11.4%) planned extubations (95% confidence interval (CI) 7.2% - 16.9%) failed. Emergency cardiac admissions (adjusted odds ratio (aOR) 7.58 (95% CI 1.90 - 30.29), dysmorphology (aOR 4.90; 95% CI 1.49 - 16.14), prematurity (aOR 4.39; 95% CI 1.24 - 15.57), and ventilation ≥48 hours (aOR 6.42 (95% CI 1.57 - 26.22) were associated with extubation failure. Children who failed extubation had longer durations of ventilation (231 hours (146.0 - 341.0) v. 53 hours (21.7 - 123.0); p<0.0001); longer duration of PICU (15 (9 - 20) days v. 5 (2 - 9) days; p<0.0001) and hospital length of stay (32 (21 - 53) days v. 15 (8 - 27) days; p=0.009); and higher 30-day mortality (28.6% v. 6.7%; p=0.001) than successfully extubated children. Conclusion: Extubation failure was associated with significant morbidity and mortality in our setting. Risk factors for extubation failure identified in our context were similar to those reported in other settings. Contributions of the study: This study provides novel data on the prevalence, risk factors and outcomes associated with extubation failure in a single-centre South African PICU. The results of this study may help identify high-risk groups for extubation failure within our local context, and forms a basis for practice improvement initiatives aimed at decreasing extubation failure rates and improving outcomes.

Effect of Cervical Lesions on Fracture Resistance and Failure Mode of Maxillary Central Incisors Restored with Fiber Posts and Complete Crowns.

PURPOSE: To investigate the effect of a cervical cavity extending 1 mm apical to the cemento-enamel junction (CEJ) on fracture resistance and failure mode of maxillary central incisors that have been treated endodontically, present with complete and incomplete ferrules, and are restored with and without a fiber post. METHODS AND MATERIALS: 50 intact human maxillary central incisors were divided into five groups (n=10): CG (control group) 6-mm fer-rule height, no cervical cavity, and without post; (CO) 6-mm ferrule height without post, with a cervical cavity (access to root canal and cervical cavity restored with composite resin), cervical cavity; and post with ferrule heights of 1 mm (CP1), 2 mm (CP2), and 6 mm (CP6) restored with fiberglass post and composite resin core. After complete metal crowns were cemented on all specimens, they were subjected to thermal cycling (6000 cycles, 5°C/55°C), followed by immediate testing of fracture resistance. After failure, the specimens were sectioned buccolingually to evaluate and identify the mode of failure. The data were analyzed with an analysis of variance (ANOVA) and the Student-Newman-Keuls multiple comparison tests (α =0.05). RESULTS: A 1-mm ferrule height (CP1) fracture resistance was significantly lower (531±125 N) compared to the 6-mm ferrule height (CP6) (769±175 N) (p<0.05). With respect to the groups with similar residual dentin, with and without a cervical cavity, CG (667±119 N) and CO (668±119 N), the presence of a post (CP6) increased the resistance to fracture, although no statistically significant difference was demonstrated. Partial decementation was observed in all specimens of CG and CP6, in nine of CP1 and CP2, and in three in CO. Root fractures occurred in 23 specimens. The root surface was exposed 2 mm below the CEJ to simulate bone level. Propagation of subosseous cracks occurred in four specimens in CG and CP2, in seven specimens in CP6, in two specimens in CP1, and in six specimens in CO. All were considered catastrophic failures. CONCLUSIONS: Within the limitations of this study it is suggested that, when restoring an endodontically treated maxillary central incisor that has a cervical lesion and needs to be restored with a complete crown, a fiber post is cemented to improve fracture resistance.

O-serotype distribution of Escherichia coli bloodstream infection isolates in critically ill patients in The Netherlands.

OBJECTIVES: Invasive infections by extra-intestinal pathogenic Escherichia coli (ExPEC) strains are increasing. We determined O-serogroups of E. coli isolates from ICU patients having bloodstream infections (BSI) and the potential coverage of a 10-valent O-polysaccharide conjugate vaccine currently in development for the prevention of invasive ExPEC disease. METHODS: We studied E. coli BSI among patients admitted to a tertiary ICU in the Netherlands between April 2011 and November 2016. O-serogroups were determined in vitro by agglutination and whole genome sequencing. RESULTS: Among 714 ICU patients having BSI, 70 (10%) had an E. coli BSI. Among 68 (97%) isolates serogrouped, the most common serogroups were O25 (n = 11; 16%), O8 (n = 5; 7%), O2 (n = 4; 6%), O6 (n = 4; 6%), and O15 (n = 4; 6%). The theoretical coverage of a 10-valent ExPEC vaccine was 54% (n = 37). CONCLUSIONS: A multi-valent ExPEC O-polysaccharide conjugate vaccine in development could potentially aid in the prevention of E. coli BSI in Dutch ICU patients.

South African guidelines on the determination of death.

Death is a medical occurrence that has social, legal, religious and cultural consequences requiring common clinical standards for its diagnosis and legal regulation. This document compiled by the Critical Care Society of Southern Africa outlines the core standards for determination of death in the hospital context. It aligns with the latest evidence-based research and international guidelines and is applicable to the South African context and legal system. The aim is to provide clear medical standards for healthcare providers to follow in the determination of death, thereby promoting safe practices and high-quality care through the use of uniform standards. Adherence to such guidelines will provide assurance to medical staff, patients, their families and the South African public that the determination of death is always undertaken with diligence, integrity, respect and compassion, and is in accordance with accepted medical standards and latest scientific evidence. The consensus guidelines were compiled using the AGREE II checklist with an 18-member expert panel participating in a three-round modified Delphi process. Checklists and advice sheets were created to assist with application of these guidelines in the clinical environment (https://criticalcare.org.za/resource/death-determination-checklists/). Key points • Brain death and circulatory death are the accepted terms for defining death in the hospital context. • Death determination is a clinical diagnosis which can be made with complete certainty provided that all preconditions are met. • The determination of death in children is held to the same standard as in adults but cannot be diagnosed in children <36 weeks' corrected gestation. • Brain-death testing while on extra-corporeal membrane oxygenation is outlined. • Recommendations are given on handling family requests for accommodation and on consideration of the potential for organ donation. • The use of a checklist combined with a rigorous testing process, comprehensive documentation and adequate counselling of the family are core tenets of death determination. This is a standard of practice to which all clinicians should adhere in end-of-life care.

South African guidelines on the determination of death.

Summary: Death is a medical occurrence that has social, legal, religious and cultural consequences requiring common clinical standards for its diagnosis and legal regulation. This document compiled by the Critical Care Society of Southern Africa outlines the core standards for determination of death in the hospital context. It aligns with the latest evidence-based research and international guidelines and is applicable to the South African context and legal system. The aim is to provide clear medical standards for healthcare providers to follow in the determination of death, thereby promoting safe practices and high-quality care through the use of uniform standards. Adherence to such guidelines will provide assurance to medical staff, patients, their families and the South African public that the determination of death is always undertaken with diligence, integrity, respect and compassion, and is in accordance with accepted medical standards and latest scientific evidence. The consensus guidelines were compiled using the AGREE II checklist with an 18-member expert panel participating in a three-round modified Delphi process. Checklists and advice sheets were created to assist with application of these guidelines in the clinical environment (https://criticalcare.org.za/resource/death-determination-checklists/). Key points: Brain death and circulatory death are the accepted terms for defining death in the hospital context.Death determination is a clinical diagnosis which can be made with complete certainty provided that all preconditions are met.The determination of death in children is held to the same standard as in adults but cannot be diagnosed in children <36 weeks' corrected gestation.Brain-death testing while on extra-corporeal membrane oxygenation is outlined.Recommendations are given on handling family requests for accommodation and on consideration of the potential for organ donation.The use of a checklist combined with a rigorous testing process, comprehensive documentation and adequate counselling of the family are core tenets of death determination. This is a standard of practice to which all clinicians should adhere in end-of-life care.

Critical care triage during the COVID-19 pandemic in South Africa: A constitutional imperative!

Triage and rationing of scarce intensive care unit (ICU) resources are an unavoidable necessity. In routine circumstances, ICU triage is premised on the best interests of an individual patient; however, when increased demand exceeds capacity, as during an infectious disease outbreak, healthcare providers need to make difficult decisions to benefit the broader community while still respecting individual interests. We are currently living through an unprecedented period, with South Africa (SA) facing the challenges of the global COVID-19 pandemic. The Critical Care Society of Southern Africa (CCSSA) expedited the development of a triage guidance document to inform the appropriate and fair use of scarce ICU resources during this pandemic. Triage decision-making is based on the clinical odds of a positive ICU outcome, balanced against the risk of mortality and longer-term morbidity affecting quality of life. Factors such as age and comorbid conditions are considered for their potential impact on clinical outcome, but are never the sole criteria for denying ICU-level care. Arbitrary, unfair discrimination is never condoned. The CCSSA COVID-19 triage guideline is aligned with SA law and international ethical standards, and upholds respect for all persons. The Bill of Rights, however, does not mandate the level of care enshrined in the constitutional right to healthcare. ICU admission is not always appropriate, available or feasible for every person suffering critical illness or injury; however, everyone has the right to receive appropriate healthcare at another level. If ICU resources are used for people who do not stand to benefit, this effectively denies others access to potentially life-saving healthcare. Appropriate triaging can therefore be considered a constitutional imperative.

In vitro effects of macrophages on orthopaedic implant alloys and local release of metallic alloy components.

AIMS: This study aimed to determine if macrophages can attach and directly affect the oxide layers of 316L stainless steel, titanium alloy (Ti6Al4V), and cobalt-chromium-molybdenum alloy (CoCrMo) by releasing components of these alloys. METHODS: Murine peritoneal macrophages were cultured and placed on stainless steel, CoCrMo, and Ti6Al4V discs into a 96-well plate. Cells were activated with interferon gamma and lipopolysaccharide. Macrophages on stainless steel discs produced significantly more nitric oxide (NO) compared to their control counterparts after eight to ten days and remained elevated for the duration of the experiment. RESULTS: On stainless steel, both nonactivated and activated cell groups were shown to have a significant increase in metal ion release for Cr, Fe, and Ni (p < 0.001, p = 0.002, and p = 0.020 respectively) compared with medium only and showed macrophage-sized corrosive pits on the stainless steel surface. On titanium alloy discs there was a significant increase in aluminum (p < 0.001) among all groups compared with medium only. CONCLUSION: These results indicated that macrophages were able to attach to and affect the oxide surface of stainless steel and titanium alloy discs. Cite this article: Bone Joint J 2020;102-B(7 Supple B):116-121.

The effect of two composite placement techniques on fracture resistance of MOD restorations with various resin composites.

OBJECTIVE: The aim of this in vitro study was to compare the effect of two restorative placement techniques, centripetal incremental technique (CIT) and bulk-fill technique (BT) on the fracture resistance of Class II MOD restorations with various resin composites in molar teeth. MATERIALS AND METHODS: Fifty-six extracted, caries free third molars were prepared with MOD preparations and restored with resin composites. The specimens were divided into two groups by placement technique, centripetal incremental technique (CIT) and bulk-fill technique (BT). Each group was subdivided into four groups according to resin composite: hybrid (Aelite LS), nano-hybrid (Virtuoso Universal), bulk fill (Filtek One Bulk Fill) and the micro-hybrid (Herculite XRV) as the control. RESULTS: Two-way analysis of variance test (ANOVA) followed by the multiple comparison procedure, Student-Newman-Keuls Method showed no a statistically significant difference between placement techniques and fracture resistance of Class II resin composite restorations (P > 0.05). Herculite XRV resisted a significantly higher load before fracture than the other three materials at a 0.05 level of significance, while Virtuoso Universal scored the lowest load. CONCLUSIONS: There was no significant effect of the two placement techniques on the fracture resistance of Class II resin composite restorations CLINICAL SIGNIFICANCE: Resin composite restorations in Class II MODs using a simplified bulk fill placement technique showed no significant difference in fracture resistance with the centripetal technique in molar teeth.

The Critical Care Society of Southern Africa guidelines on the allocation of scarce critical care resources during the COVID-19 public health emergency in South Africa.

Letter by Gopalan et al. on article by Singh and Moodley (Singh JA, Moodley K. Critical care triaging in the shadow of COVID-19: Ethics considerations. S Afr Med J 2020;110(5):355-359. https://doi.org/10.7196/SAMJ.2020.v110i5.14778); and response by Singh and Moodley.

High-frequency phenotypic switching in Candida albicans.

Most strains of Candida albicans are capable of switching spontaneously and at high frequencies between a number of phenotypes distinguished by colony morphology. Unlike switching in many other microbial pathogens, switching in C. albicans is pleiotropic, affecting several morphological and physiological parameters. Recently, the first phase-specific genes were identified and shown to be regulated at the level of gene transcription.

A pilot study of central venous catheter survival in cancer patients using low-molecular-weight heparin (dalteparin) and warfarin without catheter removal for the treatment of upper extremity deep vein thrombosis (The Catheter Study).

BACKGROUND: Central venous catheters in patients with cancer are associated with development of deep vein thrombosis (DVT); however, there is no accepted standard treatment. OBJECTIVES: To assess the safety and effectiveness of a management strategy for central venous catheter-related DVT in cancer patients consisting of dalteparin and warfarin without the need for line removal. PATIENTS/METHODS: Patients older than 18 years of age with an active malignancy and who had symptomatic, acute, objectively documented UEDVT were eligible. Patients were treated with dalteparin 200 IU kg(-1) per day for 5-7 days and warfarin with a target International Normalized Ratio of 2.0-3.0. Patients were followed for 3 months for recurrent venous thromboembolism, major hemorrhage and survival of the central venous catheter. RESULTS: There were 74 patients (48 males). The average age was 58 years. There were no episodes of recurrent venous thromboembolism and three (4%) major bleeds. No lines were removed because of infusion failure or recurrence/extension of DVT. CONCLUSION: Treatment of UEDVTs secondary to central catheters in cancer patients with standard dalteparin/warfarin can allow the central line to remain in situ with little risk of line failure or recurrence/extension of the DVT.

Prenatal cocaine exposure enhances responsivity of locus coeruleus norepinephrine neurons: role of autoreceptors.

Children exposed to cocaine during gestation have a higher incidence of neurobehavioral deficits. The neurochemical bases of these deficits have not been determined, but the pharmacology of cocaine and the nature of the abnormalities suggest that disruptions in catecholaminergic systems may be involved. In the current study, we used a rat model of prenatal cocaine exposure to examine the impact that this exposure has on the locus coeruleus (LC) noradrenergic system in offspring. Pregnant rats received twice-daily i.v. injections of cocaine (3 mg/kg) or saline between gestational days 10 and 20, and progeny were tested as juveniles. Exposure to a mild stressor elevated an index of norepinephrine turnover in the prefrontal cortex and also increased Fos expression in tyrosine hydroxylase-positive LC neurons in rats exposed to prenatal cocaine but not in rats exposed to prenatal saline. No change in the number of tyrosine hydroxylase-positive neurons in the LC was observed between the two prenatal treatment groups. Specific binding of [125I]-para-iodoclonidine, a radioligand with specificity for high affinity alpha2A-adrenergic receptors, was decreased in the LC of rats exposed to prenatal cocaine compared with prenatal saline controls. As alpha2-adrenergic receptors on LC norepinephrine neurons function as autoreceptors, their down-regulation by prenatal cocaine exposure provides a plausible mechanism for the observed heightened reactivity of norepinephrine neurons in these animals. These data indicate that prenatal cocaine exposure results in lasting changes to the regulation and responsivity of rat LC norepinephrine neurons. A similar dysregulation of LC norepinephrine neurons may occur in children exposed to cocaine during gestation, and this may explain, at least partly, the increased incidence of cognitive deficits that have been observed in these subjects.

Pregnancy intention and its relationship to birth and maternal outcomes.

OBJECTIVE: To examine whether there are associations between pregnancy intention (intended, unwanted, mistimed, or ambivalent) and negative birth and maternal outcomes: low birth weight (less than 2,500 g), preterm delivery (fewer than 37 weeks), small for gestational age, premature labor, hypertension, and other maternal outcomes. METHODS: We analyzed data from the population-based Pregnancy Risk Assessment Monitoring System, including 87,087 women who gave birth between 1996 and 1999 in 18 states. Information on pregnancy outcomes was derived from birth certificate data and a self-administered questionnaire completed postpartum. We employed SUDAAN (RTI International, Research Triangle Park, NC) for univariable and logistical regression analyses. RESULTS: In analyses controlling for demographic and behavioral factors, women with unwanted pregnancies had an increased likelihood of preterm delivery (adjusted odds ratio [OR] 1.16, 95% confidence interval [CI] 1.01-1.33) and premature rupture of membranes (adjusted OR 1.37, 95% CI 1.01-1.85) compared with women with intended pregnancies. Women who were ambivalent toward their pregnancies had increased odds of delivering a low birth weight infant (adjusted OR 1.15, 95% CI 1.02-1.29); in contrast, women with mistimed pregnancies had a lower likelihood (adjusted OR 0.92, 95% CI 0.86-0.97). CONCLUSION: Pregnancy intention, specifically unwanted and ambivalent, may be an indicator of increased risk for some poor birth and maternal outcomes and should be considered in interventions aimed at improving the health of the mother and child. LEVEL OF EVIDENCE: III.

Transcription of the gene for a pepsinogen, PEP1, is regulated by white-opaque switching in Candida albicans.

Cells of Candida albicans WO-1 spontaneously switch between a white and opaque CFU, and this phase transition involves a dramatic change in cellular phenotype. By using a differential hybridization screen, an opaque-specific cDNA, Op1a, which represents the transcript of a gene regulated by switching, has been isolated. The gene for Op1a is transcribed by opaque but not by white cells. The nucleotide sequence of the Op1a cDNA reveals over 99% base homology with an acid protease gene of C. albicans, and the predicted amino acid sequence demonstrates that the product of this gene is a member of the family of pepsinogens, which possess a hydrophobic leader sequence for secretion and two catalytic aspartate domains. Southern blots of both genomic DNA digested with 14 different endonucleases and electrophoretically separated chromosomes were probed with the Op1a cDNA. No polymorphisms were detected in either case between white and opaque cells, suggesting that no genomic reorganization occurs in the proximity of the gene during the white-opaque transition. Although transcription of Op1a correlates with the high levels of extracellular protease activity in opaque cell cultures and the absence of activity in white cell cultures, stimulation of extracellular protease activity by addition of serum albumin is not accompanied by Op1a transcription in cultures of WO-1 white cells or cultures of two additional clinical isolates of C. albicans, suggesting that expression of one or more other protease genes is stimulated in these cases. The results demonstrate that transcription of the Op1a gene is under the rigid control of switching in strain WO-1.

A bipartite DNA-binding domain in yeast Reb1p.

The REB1 gene encodes a DNA-binding protein (Reb1p) that is essential for growth of the yeast Saccharomyces cerevisiae. Reb1p binds to sites within transcriptional control regions of genes transcribed by either RNA polymerase I or RNA polymerase II. The sequence of REB1 predicts a protein of 809 amino acids. To define the DNA-binding domain of Reb1p, a series of 5' and 3' deletions within the coding region was constructed in a bacterial expression vector. Analysis of the truncated Reb1p proteins revealed that nearly 400 amino acids of the C-terminal portion of the protein are required for maximal DNA-binding activity. To further define the important structural features of Reb1p, the REB1 homolog from a related yeast, Kluyveromyces lactis, was cloned by genetic complementation. The K. lactis REB1 gene supports active growth of an S. cerevisiae strain whose REB1 gene has been deleted. The Reb1p proteins of the two organisms generate almost identical footprints on DNA, yet the K. lactis REB1 gene encodes a polypeptide of only 595 amino acids. Comparison of the two Reb1p sequences revealed that within the region necessary for the binding of Reb1p to DNA were two long regions of nearly perfect identity, separated in the S. cerevisiae Reb1p by nearly 150 amino acids but in the K. lactis Reb1p by only 40 amino acids. The first includes a 105-amino-acid region related to the DNA-binding domain of the myb oncoprotein; the second bears a faint resemblance to myb. The hypothesis that the DNA-binding domain of Reb1p is formed from these two conserved regions was confirmed by deletion of as many as 90 amino acids between them, with little effect on the DNA-binding ability of the resultant protein. We suggest that the DNA-binding domain of Reb1p is made up of two myb-like regions that, unlike myb itself, are separated by as many as 150 amino acids. Since Reb1p protects only 15 to 20 nucleotides in a chemical or enzymatic footprint assay, the protein must fold such that the two components of the binding site are adjacent.

The rRNA enhancer regulates rRNA transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the rRNA genes are organized as a tandem array of head-to-tail repeats. An enhancer of rRNA transcription is present just at the end of each transcription unit, 2 kb away from the next one. This enhancer is unusual for S. cerevisiae in that it acts both upstream and downstream of, and even across, genes. The role of the enhancer in the nutritional regulation of rRNA transcription was studied by introducing a centromere plasmid carrying two rRNA minigenes in tandem, flanking a single enhancer, into cells. Analysis of the transcripts from the two minigenes showed that the enhancer was absolutely required for the stimulation of transcription of rRNA that occurs when cells are shifted from a poor carbon source to a good carbon source. While full enhancer function is provided by a 45-bp region at the 3' end of the 190-bp enhancer, some activity was also conferred by other elements, including both a T-rich stretch and a region containing the binding sites for the proteins Reb1p and Abf1p. We conclude that the enhancer is composed of redundant elements and that it is a major element in the regulation of rRNA transcription.

REB1, a yeast DNA-binding protein with many targets, is essential for growth and bears some resemblance to the oncogene myb.

REB1 is a DNA-binding protein that recognizes sites within both the enhancer and the promoter of rRNA transcription as well as upstream of many genes transcribed by RNA polymerase II. We report here the cloning of the gene for REB1 by screening a yeast genomic lambda gt11 library with specific oligonucleotides containing the REB1 binding site consensus sequence. The REB1 gene was sequenced, revealing an open reading frame encoding 809 amino acids. The predicted protein was highly hydrophilic, with numerous OH-containing amino acids and glutamines, features common to many of the general DNA-binding proteins of Saccharomyces cerevisiae, such as ABF1, RAP1, GCN4, and HSF1. There was some homology between a portion of REB1 and the DNA-binding domain of the oncogene myb. REB1 is an essential gene that maps on chromosome II. However, the physiological role that it plays in the cell has yet to be established.