A K(ATP) channel gene effect on sleep duration: from genome-wide association studies to function in Drosophila.

Humans sleep approximately a third of their lifetime. The observation that individuals with either long or short sleep duration show associations with metabolic syndrome and psychiatric disorders suggests that the length of sleep is adaptive. Although sleep duration can be influenced by photoperiod (season) and phase of entrainment (chronotype), human familial sleep disorders indicate that there is a strong genetic modulation of sleep. Therefore, we conducted high-density genome-wide association studies for sleep duration in seven European populations (N=4251). We identified an intronic variant (rs11046205; P=3.99 × 10(-8)) in the ABCC9 gene that explains ≈5% of the variation in sleep duration. An influence of season and chronotype on sleep duration was solely observed in the replication sample (N=5949). Meta-analysis of the associations found in a subgroup of the replication sample, chosen for season of entry and chronotype, together with the discovery results showed genome-wide significance. RNA interference knockdown experiments of the conserved ABCC9 homologue in Drosophila neurons renders flies sleepless during the first 3 h of the night. ABCC9 encodes an ATP-sensitive potassium channel subunit (SUR2), serving as a sensor of intracellular energy metabolism.

Age dependent incorporation of 14C-DHA into rat brain and body tissues after dosing various 14C-DHA-esters.

INTRODUCTION: The omega-3 fatty acid docosahexaenoic acid (DHA) accounts for 10% of fatty acids in human brain and is critical for neuronal function and brain development. Mechanisms of transport, accumulation and conservation of DHA in the brain are unclear. The objective of the study was to quantify the age dependent DHA incorporation into the brain of 2-, 4- or 10-week-old rats after a bolus dose of different DHA-esters. METHODS: Rats were gavaged with (14)C-DHA-TAG, (14)C-DHA-PL or (14)C-DHA-TAG+PL at 2 mg DHA/kg BW. After 24h the distribution of radioactivity in body and brain regions was determined using quantitative whole body autoradiography (QWBA). Radiolabeled compounds were extracted from the brains to determine the identity of the radiolabeled compounds. RESULTS: Accumulation of orally ingested (14)C-DHA in rat brain was less than 1% of the dose and decreased with age. Ester specific differences were seen only in 10-week-old rats, where oral (14)C-DHA-PL delivered a 2-fold higher accretion of radioactivity in the brain. CONCLUSIONS: Less than 1% of a dietary achievable DHA dose reached the rat brain within 24h. Optimal efficacy of DHA-PL may occur in older age groups.

Detection of natural Trypanosoma vivax infections in pigs with microhaematocrit centrifugation and amplification of ITS1 rDNA.

Different species of trypanosomes may infect their mammalian hosts both singly or in combination. This study was undertaken to determine the trypanosome species that may be afflicting pigs in Uganda. Blood was collected from pigs of all ages and sexes from two districts, Kasese in Western and Jinja in Central Uganda. Of the 133 pig blood samples from Kasese that were tested for trypanosomes using the microhaematocrit centrifugation technique (MHCT), none was found to be infected. However, of the 253 pigs from Jinja district, nine were infected with trypanosomes of which three had T. vivax as determined by MHCT. However, application of the ITS1 rDNA PCR test revealed that eight pigs had T. vivax in mixed infections and one pig had T. vivax monolithic infection. These observations show that under certain circumstances, pigs may be important reservoirs for, as well as hosts to, T. vivax, contrary to earlier reports.

Preschool healthcare utilisation related to home oxygen status.

OBJECTIVE: To determine, in prematurely born children who had bronchopulmonary dysplasia (BPD), if respiratory morbidity, healthcare utilisation, and cost of care during the preschool years were influenced by use of supplementary oxygen at home after discharge from the neonatal intensive care unit. DESIGN: Observational study. SETTING: Four tertiary neonatal intensive care units. PATIENTS: 190 children, median gestational age 27 weeks (range 22-31), 70 of whom received supplementary oxygen when discharged home. INTERVENTIONS: Review of hospital and general practitioner records together with a parent completed respiratory questionnaire. MAIN OUTCOME MEASURES: Healthcare utilisation, cost of care, cough, wheeze, and use of an inhaler. RESULTS: Seventy children had supplementary oxygen at home (home oxygen group), but only one had a continuous requirement for home oxygen beyond 2 years of age. There were no significant differences in the gestational age or birth weight of the home oxygen group compared with the rest of the cohort. However, between 2 and 4 years of age inclusive, the home oxygen group had more outpatient attendances (p = 0.0021) and specialist attendances (p = 0.0023), and, for respiratory problems, required more prescriptions (p<0.0001). Their total cost of care was higher (p<0.0001). In addition, more of the home oxygen group wheezed more than once a week (p = 0.0486) and were more likely to use an inhaler (p<0.0001). CONCLUSIONS: Children with BPD who have supplementary oxygen at home after discharge have increased respiratory morbidity and healthcare utilisation in the preschool years.

Health care utilisation of prematurely born, preschool children related to hospitalisation for RSV infection.

BACKGROUND: In prematurely born infants with chronic lung disease (CLD), RSV hospitalisation is associated with increased health service utilisation and costs in the first two years after birth. AIMS: To determine whether RSV hospitalisation in the first two years was associated with chronic respiratory morbidity during the preschool years in prematurely born children who had had CLD. METHODS: Retrospective review of readmissions, outpatient attendances, and community care in years 2-4 and, at age 5 years, assessment of the children's respiratory status and their health related quality of life. Comparison was made of the results of children who had had at least one hospitalisation in the first two years after birth for RSV infection (RSV group) to those of the rest of the cohort. Participants were 190 of an original cohort of 235 infants with CLD and a median gestational age 27 (range 22-33) weeks. RESULTS: The 33 children in the RSV group, compared to the rest of the cohort, had a greater duration of hospital stay and more outpatient appointments. The RSV group had required more prescriptions for all treatments and respiratory medications, and more had used an inhaler. The cost of care of the RSV group was higher (median 2630 pounds sterling [4000 Euros, US4800 dollars], range 124-18,091 pounds sterling versus 1360 pounds sterling [2500 Euros, US3000 dollars], range 5-18 929 pounds sterling ) and their health related quality of life was lower. CONCLUSION: In prematurely born children who had developed CLD, RSV hospitalisation in the first two years was associated with chronic respiratory morbidity and increased cost of care.

Mother-to-child transmission of the human immunodeficiency virus in Texas.

BACKGROUND: The Pediatric Spectrum of HIV Diseases (PSD) project has been collecting data on HIV-exposed children in Texas since 1989. These data have now been analyzed to describe mother-to-child transmission in Texas and to provide much needed information on the magnitude of the pediatric HIV epidemic in the state. METHODS: We examined trends in the numbers of perinatally exposed children and perinatally acquired cases of HIV in the Texas PSD cohort. We calculated transmission rates and relative risks for 656 children born from January, 1995, to July, 1998, that received all or part of the ACTG 076 regimen. RESULTS: Only a small proportion (38%) of pairs of an HIV-infected mother and her HIV-exposed child received the full AIDS Clinical Trial Group 076 (ACTG 076) regimen; only 73% of the mothers received at least some prenatal care. In recent years, however, the numbers of perinatally exposed children and perinatally acquired cases of HIV have decreased in Texas. Univariate analyses showed that a reduction in the vertical transmission of HIV was associated with receipt of a full ACTG 076 regimen, receipt of a partial ACTG 076 regimen and residence in Dallas County. CONCLUSIONS: Findings identify a gap in meeting the health care needs of pregnant HIV-infected women and suggest missed opportunities to prevent mother-to-child transmission of HIV. At the same time this study confirms progress in prevention efforts to reduce mother-to-child transmission of HIV in Texas.

Parasite genome initiatives.

During 1993-1994, scientists from developing and developed countries planned and initiated a number of parasite genome projects and several consortiums for the mapping and sequencing of these medium-sized genomes were established, often based on already ongoing scientific collaborations. Financial and other support came from WHO/TDR, Wellcome Trust and other funding agencies. Thus, the genomes of Plasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi, Leishmania major, Trypanosoma brucei, Brugia malayi and other pathogenic nematodes are now under study. From an initial phase of network formation, mapping efforts and resource building (EST, GSS, phage, cosmid, BAC and YAC library constructions), sequencing was initiated in gene discovery projects but soon also on a small chromosome, and now on a fully fledged genome scale. Proteomics, functional analysis, genetic manipulation and microarray analysis are ongoing to different degrees in the respective genome initiatives, and as the funding for the whole genome sequencing becomes secured, most of the participating laboratories, apart from larger sequencing centres, become oriented to post-genomics. Bioinformatics networks are being expanded, including in developing countries, for data mining, annotation and in-depth analysis.

Spontaneous dimerization and leucine-zipper induced activation of the recombinant catalytic domain of a new adenylyl cyclase of Trypanosoma brucei, GRESAG4.4B.

In this study, we describe the isolation and characterization of a new adenylyl cyclase from Trypanosoma brucei and its activation by dimerization of the catalytic domain. In agreement with the current nomenclature of trypanosomal adenylyl cyclases, this new gene is termed GRESAG4.4B. The complete ORF of the GRESAG4.4B gene encodes a protein of 1291 amino acids. Its predicted protein structure is consistent with the structure of other trypanosomal cyclases, and with the cyclases of L. donovani. GRESAG 4.4B is constitutively expressed during the life cycle of trypanosomes. GRESAG4.4B is a member of a gene family, which contains at least six members, which are all clustered on chromosome IV. The catalytic domain of GRESAG4.4B is able to dimerize spontaneously. However, these spontaneously formed, stable dimers only show minimal enzymatic activity. The addition of a leucine zipper (LZ) derived from the S. cerevisiae GCN 4 gene to the N-terminus of the catalytic domain of GRESAG4.4B strongly activated its enzymatic activity. The LZ appears to enforce a distinct conformation of the dimer, which leads to an increased enzymatic activity, and thus may mimic the effect of ligand-induced dimerization of adenylyl cyclase in vivo.

Survival in children with perinatal HIV infection and very low CD4 lymphocyte counts.

OBJECTIVES: To evaluate clinical conditions associated with mortality in HIV-infected children with CD4+ counts <100 cells/microl. METHODS: The Pediatric Spectrum of HIV Disease Project is a longitudinal medical record review study with eight study sites in the United States, which have been enrolling children since 1989. Survival time from baseline very low CD4 count (<100 cells/microl) to death was estimated using the Kaplan-Meier method. Cox proportional hazards models were used to evaluate the effect of clinical variables on mortality. RESULTS: Of 522 children (>/=1 year of age) with serial CD4+ T-lymphocyte measurements, the median age at the first very low CD4 count was 4.8 years. The estimated median survival following the first very low CD4 count was 36 months. The following factors present at the first very low CD4 count were independently associated with a higher risk of death: younger age, weight-for-age >2 standard deviations below the mean, and previously diagnosed AIDS. The subsequent development of cytomegalovirus (CMV)-associated disease, Mycobacterium avium intracellulare (MAI) infection, wasting syndrome, or esophageal candidiasis was also independently associated with a higher risk of death. CONCLUSION: Survival in HIV-infected children with very low CD4 counts before introduction of highly active antiretroviral therapy was highly variable. Poor nutritional status and the development of CMV disease or MAI infection were associated with the shortest survival times.

Lack of definitive severe mitochondrial signs and symptoms among deceased HIV-uninfected and HIV-indeterminate children < or = 5 years of age, Pediatric Spectrum of HIV Disease project (PSD), USA.

BACKGROUND: In response to recent reports of mitochondrial dysfunction in HIV-uninfected infants exposed to antiretroviral (ARV) prophylaxis, the Perinatal Safety Review Working Group reviewed deaths in five large HIV-exposed perinatal cohorts in the United States to determine if similar cases of severe mitochondrial toxicity could be detected. We describe the results of this review for the PSD cohort. METHODS: Hospitalization, clinic and death records for deceased HIV-uninfected and HIV-indeterminate children who were less than 5 years of age were reviewed. Standard definitions were used to classify HIV infection status and the likelihood that signs and symptoms were related to mitochondrial dysfunction. Children were classified as having signs and symptoms that were considered (1) unrelated, (2) unlikely, (3) consistent with, or (4) likely related to mitochondrial disease. SIDS deaths were put into a separate category. RESULTS: 8,465 of 13,125 HIV-exposed children were either HIV-uninfected or HIV-indeterminate. Among the 84 deaths in the subgroup of 8,465 children, 9 were considered in Class 2 (unlikely), 4 were considered in Class 3 (consistent with), and none were considered in Class 4 (likely). 97% of those children who received ARV prophylaxis received zidovudine alone. None of the HIV-uninfected deaths were classified in 2, 3, or 4; and only one of these was exposed to ARV prophylaxis. Among the 3 HIV-indeterminate children who were classified in 3 (consistent with), 2 had no or unknown ARV exposure before 1994 when use of ZDV prophylaxis became the standard of care. Both HIV-uninfected and HIV-indeterminate children with ARV exposure or unknown exposure had lower mortality rates than children without ARV exposure. CONCLUSION: Monoprophylaxis with ZDV was not associated with higher death rates in the cohort of 8,465 children or with any findings likely consistent with mitochondrial dysfunction among the 85 deaths. Ongoing monitoring of drug safety in large multi-site prospective cohort studies of HIV-exposed children is essential in the era of highly active antiretroviral therapy.

The African trypanosome genome.

The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite.

The molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427.

We present the molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock 427, clone 221a. This cloned stock is most commonly used in research laboratories in genetic manipulation experiments and in studies of antigenic variation. Using 116 previously characterised chromosome-specific markers, we identify 11 diploid pairs of megabase chromosomes and detect no loss of synteny in EST and gene marker distribution between this stock and the genome project reference stock TREU 927/4. Nevertheless, the chromosomes of 427 are all larger than their homologues in 927, except chromosomes IIa and IXa. The greatest size variation is seen in chromosome I, the smallest of which is 1.1 Mb (927-Ia) and the largest 3.6 Mb (427-Ib). The total nuclear DNA content of both stocks has been estimated by comparison of the mobility of T. brucei and yeast chromosomes. Trypanosomes of stock 427 contain approximately 16.5 Mb more megabase chromosomal DNA than those of stock 927. We have detected the presence of bloodstream-form expression-site-associated sequences on eight or more megabase chromosomes. These sequences are not found on the same chromosomes in each stock. We have determined the chromosomal band location of nine characterised variant surface glycoprotein genes, including the currently expressed VSG 221. Our results demonstrate both the stability of the T. brucei genome, as illustrated by the conservation of syntenic groups of genes in the two stocks, and the polymorphic nature of the genomic regions involved in antigenic variation. We propose that the chromosomes of stock 427 be numbered to correspond to their homologues in the genome project reference stock TREU 927/4.

The anaerobic pathogen Clostridium perfringens can escape the phagosome of macrophages under aerobic conditions.

Clostridium perfringens is the most common cause of gas gangrene (clostridial myonecrosis), a disease that begins when ischaemic tissues become contaminated with C. perfringens vegetative cells or spores. An aerotolerant anaerobe, C. perfringens quickly multiplies in ischaemic tissues and spreads to healthy areas, leading to a high level of morbidity and mortality. As a species, the bacterium can synthesize 13 different toxins, and these are thought to be the major virulence factors of the disease. However, we present evidence here that C. perfringens can also persist inside macrophages, under aerobic conditions, by escaping the phagosome into the cytoplasm. C. perfringens was not killed by the cells of a clone (J774-33) of the macrophage-like murine cell line J774A.1 under aerobic or anaerobic conditions, whereas the non-pathogenic bacterium Bacillus subtilis was killed by J774-33 cells under both conditions. Electron microscopy images showed that C. perfringens cells were intact and resided mostly in the cytoplasm of J774-33 cells, whereas B. subtilis was in the phagosome. Immunofluorescence microscopy showed that intracellular C. perfringens bacteria failed to co-localize with the late endosome-lysosomal marker glycoprotein LAMP-1, whereas B. subtilis did co-localize with LAMP-1. C. perfringens also appeared to escape the phagosome of both activated and unactivated mouse peritoneal macrophages, but not as efficiently as was seen with the J774-33 cell line. In addition, cytochalasin D was used to show that phagocytosis of C. perfringens was dependent on actin polymerization and that the bacteria attach to J774-33 cells at distinct areas of the cell membrane. We propose that the ability to escape the phagosome and persist inside macrophages is an important factor in the early stages of a gangrene infection, when bacterial numbers are low and phagocytic cells are present.

Analysis of ploidy (in megabase chromosomes) in Trypanosoma brucei after genetic exchange.

The megabase chromosomes of Trypanosoma brucei are normally diploid, but the extent to which this ploidy is maintained when parasites undergo genetic exchange is not known. To investigate this issue, a panel of 30 recombinant clones resulting from the co-transmission through tsetse flies of three different parental T. brucei lines in all pair-wise combinations (STIB 247, STIB 386 and TREU 927/4) were examined. These clones are products of 28 different mating events; four of them result from self-fertilisation and the others are F1 hybrids. DNA contents of the three parental lines were determined by flow cytometry and shown to differ only slightly with DNA content increasing in the order 927/4 < 247 < 386. Flow cytometry of the recombinant clones indicated DNA contents were similar to the parents in 28 clones and raised approximately 1.5 times the parental values in only two. The two F1 hybrid progeny with raised DNA contents were shown by marker analysis to be trisomic for seven independent loci indicating that they were probably triploid whereas progeny with DNA contents similar to parental values inherited a single allele from each parent for four independent loci indicating that they were diploid.

Health care providers' perspectives on patient delay for seeking care for symptoms of acute myocardial infarction.

To inform intervention development in a multisite randomized community trial, the Rapid Early Action for Coronary Treatment (REACT) project formative research was undertaken for the purpose of investigating the knowledge, beliefs, perceptions, and usual practice of health care professionals. A total of 24 key informant interviews of cardiologists and emergency physicians and 15 focus groups (91 participants) were conducted in five major geographic regions: Northeast, Northwest, Southeast, Southwest, and Midwest. Transcript analyses revealed that clinicians are somewhat unaware of the empirical evidence related to the problem of patient delay, are concerned about the practice constraints they face, and would benefit from concrete suggestions about how to improve patient education and encourage fast action. Findings provide guidance for selection of educational strategies and messages for health providers as well as patients and the public.

Status of protozoan genome analysis: trypanosomatids.

The three trypanosomatid genome projects have employed common strategies which include: analysis of pulsed-field gel electrophoretic chromosomal karyotypes; physical mapping using big DNA (cosmid, pacmid P1, bacterial artificial chromosome, yeast artificial chromosome) libraries; partial cDNA sequence analysis to develop sets of expressed sequence tags (ESTs) for gene discovery and use as markers in physical mapping; genomic sequencing; dissemination of information through development of web-sites and ACeDB-based fully integrated databases; and establishment of functional genomics programmes to maximize useful application of genome data. Highlights of the projects to date have been the demonstration that, despite extensive chromosomal size polymorphisms for diploid homologues within Africa trypanosomes, T. cruzi or Leishmania, the physical linkage groups for markers on each chromosome are retained across all isolates/species studied within each group. For African trypanosomes, detailed analysis of chromosome 1 has demonstrated that repetitive sequences and the two retroposon-like elements RIME and INGI are localized to a defined region at one end of the chromosome, with the bulk of the central region of the chromosome containing genes coding for expressed proteins. Comparative mapping shows that, although subtelomeric changes account for a large proportion of the polymorphism in chromosome size in African trypanosomes, there are significant expansions and contractions in regions across the entire chromosome. The highlight of the genomic sequencing projects has been the demonstration of just 2 putative transcriptional units of chromosome 1 of Leishmania major, extending on opposite strands from a point in the central region of the chromosome. A similar observation made on 93.4 kb of contiguous sequence for T. cruzi chromosome 3 suggests the presence of promoter and regulatory elements at the junctions of large polycistronic transcriptional units. All data obtained from the genome projects are made available through the public domain, which has prompted changing philosophies in how we approach analysis of the biology of these organisms, and strategies that we can employ now in the search for new therapies and vaccines.

Predominance of duplicative VSG gene conversion in antigenic variation in African trypanosomes.

A number of mechanisms have been described by which African trypanosomes undergo the genetic switches that differentially activate their variant surface glycoprotein genes (VSGs) and bring about antigenic variation. These mechanisms have been observed mainly in trypanosome lines adapted, by rapid syringe passaging, to laboratory conditions. Such "monomorphic" lines, which routinely yield only the proliferative bloodstream form and do not develop through their life cycle, have VSG switch rates up to 4 or 5 orders of magnitude lower than those of nonadapted lines. We have proposed that nonadapted, or pleomorphic, trypanosomes normally have an active VSG switch mechanism, involving gene duplication, that is depressed, or from which a component is absent, in monomorphic lines. We have characterized 88 trypanosome clones from the first two relapse peaks of a single rabbit infection with pleomorphic trypanosomes and shown that they represent 11 different variable antigen types (VATs). The pattern of appearance in the first relapse peak was generally reproducible in three more rabbit infections. Nine of these VATs had activated VSGs by gene duplication, the tenth possibly also had done so, and only one had activated a VSG by the transcriptional switch mechanism that predominates in monomorphic lines. At least 10 of the donor genes have telomeric silent copies, and many reside on minichromosomes. It appears that trypanosome antigenic variation is dominated by one, relatively highly active, mechanism rather than by the plethora of pathways described before.

Cloning, sequence, and transcriptional regulation of the operon encoding a putative N-acetylmannosamine-6-phosphate epimerase (nanE) and sialic acid lyase (nanA) in Clostridium perfringens.

Clostridium perfringens can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates. After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and N-acetylmannosamine. The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts N-acetylmannosamine-6-phosphate to N-acetylglucosamine-6-phosphate. A 4.0-kb DNA fragment from C. perfringens NCTC 8798 that contains the nanE and nanA genes has been cloned. The identification of the nanA gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a nanA Escherichia coli strain, EV78. The nanA gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source. By using Northern blot experiments, it was demonstrated that the nanE and nanA genes comprise an operon and that transcription of the operon in C. perfringens is inducible by the addition of sialic acid to the growth medium. The Northern blot experiments also showed that there is no catabolite repression of nanE-nanA transcription by glucose. With a plasmid construct containing a promoterless cpe-gusA gene fusion, in which beta-glucuronidase activity indicated that the gusA gene acted as a reporter for transcription, a promoter was localized to the region upstream of the nanE gene. Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the nanE coding sequence.

Multiple causes of size variation in the diploid megabase chromosomes of African tyrpanosomes.

The chromosomes of many protozoans are polymorphic in size, but African trypanosomes contain diploid homologues which are exceptionally size-polymorphic. We present the first complete analysis of the structure of a Trypanosoma brucei megabase chromosome which reveals the concentration of repetitive sequence, non-random distribution of transposon-like elements, and a hemizygous variant surface glycoprotein gene expression site. Subsequent comparative analyses of size-polymorphic homologues show that the repetitive regions are highly polymorphic, as demonstrated in studies on the chromosomes of other protozoan parasites. We show that a large number of the transposon-like elements are located in these regions. However, although we have shown elsewhere that synteny is maintained in coding regions, homologous chromosomes may vary along their entire length. Thus, the variable chromosomal location of variant surface glycoprotein expression gene sites, the expansion and contraction of repetitive DNA, the number of putative transposons, sequence polymorphism at chromosome ends, and expansion and contraction within or between coding regions all contribute to huge chromosomal size polymorphisms in T brucei.

Transcription of 'inactive' expression sites in African trypanosomes leads to expression of multiple transferrin receptor RNAs in bloodstream forms.

African trypanosomes express a heterodimeric transferrin receptor that mediates iron uptake from the host bloodstream. The genes encoding the receptor, ESAG6 and ESAG7, are found at the beginning of VSG expression sites: these are telomeric, polycistronic transcription units that each terminate with a gene encoding a trypanosome variant surface glycoprotein, VSG. Approximately 20 of these VSG expression sites are found in the trypanosome genome, but only one VSG is expressed at a time. The conventional view is that one expression site promoter is extremely active whereas the others are either inactive or show very low, poorly processive activity, and that all transferrin receptor molecules are encoded by the active expression site. The 3'-end of the ESAG6 gene is more than 5 kb from the promoter. We show here that 20% of ESAG6 mRNA originates from the 'inactive' expression sites. We suggest that many expression site promoters in trypanosomes show low-level activity throughout the life cycle, and that transcription proceeds for at least 5 kb. This suggests a simplified model of VSG expression site control, whereby the only regulated event is the strong activation of a single expression site promoter in bloodstream forms.