Raltegravir thorough QT/QTc study: a single supratherapeutic dose of raltegravir does not prolong the QTcF interval.

Raltegravir is a novel HIV-1 integrase inhibitor with potent in vitro activity (IC(95) = 31 nM in 50% human serum). A double-blind, randomized, placebo-controlled, double-dummy, 3-period, single-dose crossover study was conducted; subjects received single oral doses of 1600 mg raltegravir, 400 mg moxifloxacin, and placebo. The upper limit of the 2-sided 90% confidence interval for the QTcF interval placebo-adjusted mean change from baseline of raltegravir was less than 10 ms at every time point. For the raltegravir and placebo groups, there were no QTcF values >450 ms or change from baseline values >30 ms. A mean C(max) of approximately 20 muM raltegravir was attained, approximately 4-fold higher than the C(max) at the clinical dose. Moxifloxacin demonstrated an increase in QTcF at the 2-, 3-, and 4-hour time points. Administration of a single supratherapeutic dose of raltegravir does not prolong the QTcF interval. A single supratherapeutic dose design may be appropriate for crossover thorough QTc studies.

A proposed mechanism for promoter-associated DNA rearrangement events at a variant surface glycoprotein gene expression site.

The expressed variant cell surface glycoprotein (VSG) gene of the protozoan parasite Trypanosoma brucei is invariably found at one of several telomeric VSG gene expression sites (ESs). The active ES in variant 118 clone 1 is found on a 1.5-Mb chromosome, and the promoter region is located more than 45 kb upstream of the VSG gene. We had previously shown that DNA rearrangement events occurred in the promoter region, specifically at inactivation of this ES (K. M. Gottesdiener, H.-M. Chung, S. L. Brown, M. G.-S. Lee, and L. H. T. Van der Ploeg, Mol. Cell. Biol. 11:2467-2477, 1991). In this report, we describe the cloning of the entire 17-kb promoter region, which revealed the presence of two identical 2.15-kb tandem promoter repeats separated by 13 kb of DNA. The two virtually identical promoter repeats both function efficiently in directing transcription in transient transfection assays in insect-form trypanosomes. We characterized the DNA rearrangement events that occur at ES inactivation, and by studying both of the reciprocal products of this recombination event, we infer that these result from direct (promoter) repeat recombination, formation of heteroduplex DNA, and a reciprocal exchange event that releases a circular DNA as a side product of the reaction. The finding of DNA recombinational events in a region of the VSG gene ES that encodes the promoter(s), and their relatively frequent occurrence at ES inactivation, suggests a possible role in ES control.

Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

Individual and combined effects of peroxisome proliferator-activated receptor and {gamma} agonists, fenofibrate and rosiglitazone, on biomarkers of lipid and glucose metabolism in healthy nondiabetic volunteers.

This open-label, randomized, placebo-controlled, incomplete-block, 3-period crossover pilot study investigated the effects of peroxisome proliferator-activated receptor alpha- and gamma-agonists on biomarkers of lipid and glucose metabolism in 12 nondiabetic subjects. Plasma samples were collected before and after each 14-day treatment with placebo, fenofibrate (201 mg/d), rosiglitazone (4 mg twice daily), and combined fenofibrate (201 mg/d) plus rosiglitazone (4 mg twice daily). Except for triglycerides (P < .042) and free fatty acids (P < .074), no significant interaction was demonstrated between fenofibrate and rosiglitazone; thus, the effect due to each drug alone was evaluated (presence/absence of drug). Fenofibrate significantly (P < .050) increased lipoprotein lipase activity (35%) and decreased apolipoproteins B (13%) and C-III (20%). Rosiglitazone significantly (P < .050) decreased fasting glucose (7.3%) and increased apolipoprotein C-III (19%) and adiponectin (137%). Fenofibrate and rosiglitazone also produced effects on triglycerides and free fatty acids, but it was not possible to determine if these effects were synergistic in nature.

Comparison of rofecoxib, celecoxib, and naproxen on renal function in elderly subjects receiving a normal-salt diet.

BACKGROUND: This study compared directly the renal effects of two selective cyclooxygenase (COX)-2 inhibitors (rofecoxib and celecoxib) with naproxen (dual COX-1/COX-2 inhibitor) and placebo in healthy elderly subjects on a sodium-replete diet. METHODS: A total of 67 elderly subjects stabilized in the clinic for weight and urinary sodium on a controlled 200-mEq sodium diet were randomized in a double-blind fashion to receive rofecoxib, 25 mg daily (n = 17); celecoxib, 200 mg twice daily (n = 17); naproxen, 500 mg twice daily (n = 17); or matching placebo (n = 16) for 28 days. Subjects were sequestered in the clinic for the first 14 treatment days on the controlled diet. RESULTS: Daily urinary sodium excretion during the first 72 hours of treatment (primary endpoint) significantly decreased in rofecoxib, celecoxib, and naproxen groups compared with baseline (P < or =.05). Rofecoxib and celecoxib decreases in urinary sodium excretion rates that were comparable with each other, on the basis of predefined boundaries (-39.5 versus -27.1 mEq/d, respectively) and to naproxen (-40.6, mEq/d). Rofecoxib, celecoxib, and naproxen increased mean systolic blood pressure to a similar degree (3.4, 4.3, and 3.1 mm Hg, respectively, versus -1.3 mm Hg for placebo) after 14 days of treatment; small changes also occurred in diastolic blood pressure (0.3, 0.8, and -0.4 mm Hg, respectively, versus -1.4 mm Hg for placebo). Changes from baseline in creatinine clearance, body weight, and urinary potassium excretion among active treatments were similar. After 28 days of treatment, findings were generally consistent with those at 14 days. No subject reported edema or discontinued treatment as the result of an adverse experience. CONCLUSION: In healthy elderly subjects on a sodium-replete diet, the COX-2 inhibitors rofecoxib and celecoxib did not differ from a nonselective nonsteroidal anti-inflammatory drug (naproxen), in influencing renal function as measured by urinary sodium excretion, systolic and diastolic blood pressure, creatinine clearance, or weight change.

A new VSG expression site-associated gene (ESAG) in the promoter region of Trypanosoma brucei encodes a protein with 10 potential transmembrane domains.

In Trypanosoma brucei bloodstream variants 118 cl 1, 118a and 118b, the actively transcribed VSG gene expression site (ES) is located on a 1.5 Mb chromosome. The promoter region for this polycistronic transcription unit is unusual in that there are two, tandemly located, promoter repeats, each 2.1 kb in size, separated by 13 kb of intervening DNA. As previously shown, at inactivation of this ES, the promoter region was rearranged with the deletion of 15 kb of DNA. This result prompted us to search through the deleted DNA sequences to identify additional genes that might play a role in the inactivation of ESs. In this report, we identify a gene, encoding a putative transmembrane protein, that was deleted at this locus by the rearrangement event. This gene, which we tentatively call expression-site-associated-gene 10 (ESAG10), contains 10 potential transmembrane domains and had been located to T. brucei stock 427-60, ES-containing chromosomes.

Analysis of a hybrid PARP/VSG ES promoter in procyclic trypanosomes.

The parasite Trypanosoma brucei changes its variant surface glycoprotein (VSG) coat to escape the host immune system. At a chromosomal locus, we analyzed the promoter that controls expression of VSG genes, using a system developed in collaboration with Urményi and Van der Ploeg (Urményi, T.P. and Van der Ploeg, L.H.T. (1995) Nucleic Acids Res. 23,1010-1016), and showed that the variant surface glycoprotein expression site (VSG ES) promoter directed < 6% the CAT activity produced by the procyclic acidic repetitive protein (PARP) promoter at the same locus. We identified a fragment from the PARP promoter (bp -743 to -111) that contained no intrinsic promoter activity. However, when this fragment was cloned 5' to 3' upstream of the VSG ES promoter, and this hybrid PARP/VSG ES promoter was stably integrated at the RNA polymerase (Pol) II largest subunit gene locus, expression from a CAT gene cassette increased 10-fold. Nascent RNA analysis independently showed that the relative efficiency of alpha-amanitin-resistant transcription directed by the hybrid PARP/VSG ES promoter was more than 6-fold higher than that directed by the wild-type VSG ES promoter. Furthermore, using nascent RNA protection assays, we mapped the transcription start site of the hybrid PARP/VSG ES promoter to the same initiation site as that of the wild-type VSG ES promoter. Finally, we evaluated the functional activity of the hybrid PARP/VSG ES mutant promoter at the dominant VSG gene expression site on the 1.5-Mb chromosome. At this locus, as well, the hybrid PARP/VSG ES promoter directed almost 3-times as much CAT activity as that of the wild-type VSG ES promoter.

Procyclic Trypanosoma brucei cell lines deficient in ornithine decarboxylase activity.

Ornithine decarboxylase (ODC) is a rate limiting enzyme in the biosynthesis of polyamines. We report here the construction of ODC gene deficient Trypanosoma brucei brucei cell lines by homologous recombination and disruption of the two alleles of the ODC gene. With our first stable transfection vector, we replaced the 2.8 kb SacII ODC gene-containing fragment with a hygromycin-B-phosphotransferase gene (hph) cassette transcribed under the control of the endogenous promoter. For the second ODC allele knock-out, we stably transfected similar constructs that contained either the phleomycin or G418 resistance gene cassette, and included 1 mM putrescine in the media. These experiments resulted in two separate ODC- lines: one hygromycin and phleomycin resistant, the other hygromycin and G418 resistant. The two ODC gene knockout lines were verified by Southern and Northern hybridization, and confirmed by Western blot and enzymatic activity assay. There is no ODC expression in the two ODC- lines and the ODC messages in the single ODC gene knockouts were only half of that of the wild type. When grown in the presence of putrescine, the ODC- lines showed little difference, morphologically, from wild type trypanosomes. The growth rate of these lines varied greatly, depending on the concentration of the putrescine. Interestingly, when putrescine was completely withdrawn from the media, the ODC- trypanosomes soon reached a plateau phase and some cells remained viable for 7-8 weeks. The starved cells could be rescued by the addition of putrescine or introducing back the ODC gene. Cell cycle analysis suggested that putrescine is required for G1-S transition in the procyclic form T. brucei.

A detailed mutational analysis of the VSG gene expression site promoter.

The African trypanosome Trypanosoma brucei is a protozoan parasite that causes the disease African sleeping sickness. The parasite avoids the host's immune response by the process of antigenic variation, or by sequentially expressing antigenically different cell-surface coat proteins. These proteins, called variant surface glycoproteins (VSGs), are expressed from a specific locus, the VSG gene expression site (ES). In an attempt to understand expression of VSG genes, we expanded on earlier investigations of the promoter that controls the large VSG gene expression site transcription unit. We studied VSG ES promoter function both in transient transfection assays, and after stable integration at a chromosomal locus. Analysis of closely spaced deletion mutants showed that the minimum VSG ES promoter fragment that gives full activity is extremely small, and mapped precisely to a fragment that contains no more than -67 bp 5' to the putative transcription initiation site. The promoter lacked an upstream control element, or UCE, an element found at the PARP promoter, and at most eukaryotic Pol I promoters. Furthermore, linker scanning mutagenesis demonstrated that the VSG ES promoter contains at least two essential regulatory elements, including sequences within the region -67/-60 and the region -35/-20, both numbered relative to the initiation site. An altered promoter with mutated nucleotides surrounding the transcription initiation site still directed wild-type levels of expression. In this study, the results were similar for both insect and bloodstream form trypanosomes, suggesting that the same basic machinery for expression from the VSG ES promoter is found in both stages of the parasite.

Binding of trans-acting factors to the double-stranded variant surface glycoprotein (VSG) expression site promoter of Trypanosoma brucei.

Trypanosoma brucei evades its host's immune response by utilizing the system of antigenic variation, whereby the organism sequentially expresses antigenically distinct variant surface glycoproteins (VSGs). Actively expressed VSG genes are found in VSG expression sites (ESs), and transcription of these ESs is directed by a small promoter composed of two essential cis-acting elements, the VSG ES promoter upstream element (VUE) and VSG ES promoter downstream element (VDE). Using electrophoretic mobility shift assays, we have identified double-stranded DNA binding activity in bloodstream-form trypanosome nuclear extracts. This activity, the VEP complex, is specific for the VSG ES promoter, and requires the intact sequences of the VUE and VDE in the appropriate spacing. These requirements of VEP Complex formation parallel the requirements for promoter function, suggesting that the VEP complex may be composed of functionally significant trans-acting factors. Furthermore, the requirement of both elements suggests that the binding of factors to the promoter may be cooperative. However, subtly different binding characteristics were observed when we used nuclear extracts derived from procyclic trypanosomes.

Cloning by functional complementation in Trypanosoma brucei.

A procyclic Trypanosoma brucei double-knockout mutant lacking the ornithine decarboxylase (ODC) gene was transfected with a T. brucei genomic library in the expression vector pTSO-HYG4, which utilizes the PARP promoter and replicates extrachromosomally by virtue of a minicircle origin of replication. Transfectants which grew in the absence of exogenous putrescine, the product of the ODC-catalyzed reaction, were obtained at a frequency of 1.6 x 10(-7) and shown to restore ODC protein synthesis and enzymatic activity. Restriction enzyme patterns and Southern blot analysis of plasmids recovered from these cells and propagated in E. coli showed that the inserts contained a single copy of the T. brucei ODC gene. These results demonstrate for the first time the feasibility of identifying novel T. brucei genes by direct complementation of mutant T. brucei cell lines.

Dose proportionality of oral etoricoxib, a highly selective cyclooxygenase-2 inhibitor, in healthy volunteers.

To assess dose proportionality of etoricoxib across the anticipated clinical dose range, a single panel of 12 healthy subjects was administered single oral doses of etoricoxib of 5, 10, 20, 40, and 120 mg in an open, two-part, five-period crossover study. Plasma samples were collected aftereach dose and analyzed for etoricoxib concentrations. The pharmacokinetics of etoricoxib appear to be linear over the entire dose range examined, from 5 to 120 mg. Etoricoxib was found to be well tolerated across the 5 to 120 mg dose range.

Protozoan genomes : karyotype analysis, chromosome structure, and chromosome specific libraries.

Protozoa represent a diverse group of single-celled eukaryotes, many of which have parasitic life styles, infecting hundreds of millions of people. Unique aspects of their biology relate to their distinct evolutionary position and their complex life cycles, frequently involving different hosts.

The effects of TPA023, a GABAAα2,3 subtype-selective partial agonist, on essential tremor in comparison to alcohol.

Essential tremor (ET) is a relatively frequent neurological disorder that responds in some patients to gamma-aminobutyric acid A (GABA(A)) agonists such as the benzodiazepines. Partial subtype-selective GABA(A) agonists may have an improved side effect profile compared to non-selective GABA(A) agonists. However, it is unknown which GABA(A) subtypes are involved in the therapeutic effects of benzodiazepines in ET. The effects of 2 mg TPA023, a GABA(A) α2,3 subtype-selective partial agonist, on ET were compared to the effects of a stable alcohol level (0.6 g/L) and placebo in nine patients with ET. Tremor evaluation included laboratory accelerometry and a performance-based scale. Additional measurements were performed to evaluate other effects on the central nervous system (CNS). Alcohol significantly diminished tremor symptoms in the postural and kinetic condition, as assessed by laboratory accelerometry, but the performance-based rating scale was unaffected. Tremor was also reduced after TPA023 treatment in the kinetic condition, albeit not significantly. Additionally, TPA023 decreased saccadic peak velocity, while alcohol decreased subjective feelings of alertness. This study showed that alcohol reduced maximum tremor power, as assessed by laboratory accelerometry, unlike TPA023, which decreased tremor symptoms to some extent but not significantly. This study showed that treatment with an α2,3 subunit-selective GABA(A) partial agonist was less effective than a stable level of alcohol in reducing ET symptoms. These results provide no support for a therapeutic role of TPA023 in the suppression of ET symptoms.

Safety, tolerability, and pharmacokinetics of raltegravir after single and multiple doses in healthy subjects.

Raltegravir is a novel human immunodeficiency virus-1 integrase inhibitor with potent in vitro activity (95% inhibitory concentration (IC95)=33 nM in 50% human serum). Three double-blind, randomized, placebo-controlled, pharmacokinetic, safety, and tolerability studies were conducted: (1) single-dose escalation study (10-1,600 mg), (2) multiple-dose escalation study (100-800 mg q12 h x 10 days), and (3) single-dose female study (400 mg). Raltegravir was rapidly absorbed with a terminal half-life (t1/2) approximately 7-12 h. Approximately 7-14% of raltegravir was excreted unchanged in urine. Area under the curve (AUC)(0-infinity) was similar between male and female subjects. After multiple-dose administration, steady state was achieved within 2 days; there was little to modest accumulation of raltegravir. Trough levels were >33 nM for dose levels of 100 mg and greater. Raltegravir is generally well tolerated at doses of up to 1,600 mg/day given for up to 10 days and exhibits a pharmacokinetic profile supportive of twice-daily dosing with multiple doses of 100 mg and greater achieving trough levels >33 nM.

The effect of MK-0524, a prostaglandin D(2) receptor antagonist, on prostaglandin D (2)-induced nasal airway obstruction in healthy volunteers.

INTRODUCTION: Nasal congestion in allergic rhinitis results from tissue edema and vasodilatation in the nasal mucosa. Of the mediators released by mast cells in response to allergens, prostaglandin (PG) D(2) is regarded as the most potent inducer of nasal congestion. Intranasal administration of PGD(2) reproduces the nasal blockade experienced by patients with seasonal allergic rhinitis (SAR) via its action on the PGD(2) (DP) receptor to induce nasal vasodilatation. Intranasal challenge with PGD(2) can be a useful tool for evaluating DP-receptor antagonists. OBJECTIVE: The main purpose of this study was to examine the ability of MK-0524, a DP receptor antagonist in development for the treatment of SAR, to block PGD(2) induced nasal congestion in healthy volunteers. METHODS: To this end, a double-blind, placebo-controlled, randomized, 3-period study was performed in 15 healthy subjects. During each period, subjects received MK-0524 25 mg, MK-0524 100 mg or placebo qd for 3 days. Twenty-four hours following the last dose, nasal provocations with PGD(2) were performed to determine the PD(75), which is the intranasal dose of PGD(2) that provokes a 75% increase in baseline total nasal airway resistance as performed by active anterior rhinomanometry. RESULTS: Following treatment with MK-0524, the PD(75) (mean+/-SD) was significantly shifted from 15.8 +/- 18.3 mug/nostril during the placebo period to more than 512 mug/nostril both following the 25- and 100-mg (maximum challenge dose tested) dose regimen. CONCLUSION: Whether this >45 fold increase in PD(75) will induce a clinically meaningful effect of MK-0524 will require clinical study in participants with SAR.

Transplanted infections: donor-to-host transmission with the allograft.

PURPOSE: To evaluate the transmission of infectious agents from organ donors to transplant recipients, and to assess risk factors for transmission, primarily in recipients of kidney, cornea, and heart allografts. DATA IDENTIFICATION: Computerized literature searches of MEDLINE and PAPERCHASE through January 1988, extensive review of references from identified articles, and review of major clinical and transplantation journals through June 1988. STUDY SELECTION: All case reports and studies that reported a possible donor-to-recipient transmission of infection were selected and reviewed. DATA EXTRACTION: Each case report or patient series of donor-to-recipient transmission was judged as possible, probable, or proven depending on the completeness of donor and recipient information available and the likelihood of alternate causes of infection. RESULTS OF DATA SYNTHESIS: True donor-transmitted infection can occur with viruses including human immunodeficiency virus, cytomegalovirus, herpes simplex, Epstein-Barr, rabies, the virus causing Creutzfeldt-Jakob disease, and with hepatitis B virus. It can also occur with many common aerobic bacteria, although allograft-transmitted bacterial infection is more often caused by contamination during harvesting and processing. Fungi and yeast, as well as toxoplasmosis, have been transmitted less frequently, and there have been rare instances when mycobacterial infection, malaria, trypanosomiasis, and strongyloidiasis have been transplanted with the donor organ. CONCLUSIONS: Infection can be transmitted with a donor organ to the recipient, but contamination of the organ during processing and harvesting is commoner and may lead to severe infection in the recipient, especially if contamination is by one of a subset of more virulent organisms. True donor-transmitted infection, although rare, can be reduced by careful donor screening, which should include clinical and epidemiologic assessment for evidence of infection, as well as judicious laboratory testing.

A Trypanosoma brucei bloodstream form mutant deficient in ornithine decarboxylase can protect against wild-type infection in mice.

A Trypanosoma brucei bloodstream mutant in which both copies of the ornithine decarboxylase (ODC) gene were knocked out (ODC mutant) was used to determine the biological functions of ODC in T. brucei. Growth of the mutant cells ceased within 12-24 h in regular culture medium deficient in polyamines, but could be rescued by supplementation with 1 mM putrescine. A mouse model of T. brucei infection was used to determine whether the mutant was still infective and was found to develop either extremely low or undetectable levels of parasitemia, suggesting that in T. brucei, ODC activity is essential for establishing an infection. Furthermore, when these mice were subsequently challenged with wild-type T. brucei cells expressing the same variant surface glycoprotein (VSG), they did not develop any parasitemia, indicating that inoculating the mice with the attenuated ODC mutant had conferred protection against challenge by wild-type cells. These results were reproduced in C57BL/6J mice deficient in alpha-beta and gamma-delta T-cell receptors. However, no protection was observed in rag-2 knockout mice deficient in both B and T lymphocytes or in C57BL/10J mice deficient only in B lymphocytes. The results thus suggest that the ODC mutant could induce a T-lymphocyte-independent but B-lymphocyte-dependent immunity against wild-type cells of the same VSG. Such a mechanism of immunity has been elicited only by live T. brucei cells, but not by isolated VSGs or radiation-killed trypanosomes. This ODC mutant may thus represent a genuinely attenuated T. brucei bloodstream form capable of immunizing mammals against infections by African trypanosomes of the same VSG subtype without causing detectable infection by itself. The observation also raises the interesting likelihood that the in vivo treatment of T. brucei bloodstream forms with alpha-DL-difluoromethylornithine is a de facto attenuation of the parasitic organisms, which may very well result in B-lymphocyte-dependent host immune responses to subsequent infections by parasites of the same VSG subtypes.

Trypanosoma brucei brucei: characterization of an ODC null bloodstream form mutant and the action of alpha-difluoromethylornithine.

Ornithine decarboxylase (ODC) is a key enzyme in the polyamine synthesis pathway in African trypanosomes. We report here the characterization of an ODC null bloodstream form Trypanosoma brucei brucei mutant, created by replacing the ODC gene with antibiotic resistant marker genes through transfection and homologous recombination. The null mutant expresses no ODC mRNA or protein and does not have ODC enzymatic activity. We tested the attenuation of the bloodstream form ODC- mutants in mice, and showed that these mutants cannot multiply and are quickly cleared from the blood. We also tested the effect of DFMO on this ODC null mutant.

Improved separation of chromosome-sized DNA from Trypanosoma brucei, stock 427-60.

Separation of chromosome-sized DNA from the parasitic protozoan Trypanosoma brucei had previously resulted in the fractionation of DNA molecules that ranged in size from 50 kb up to roughly 1.5 Mb. The number of larger chromosomes and their size, accounting for 80% of the DNA of T. brucei remained unclear. We have now size separated these larger DNA molecules by pulsed field gel electrophoresis (PFG) and resolve a total of 20 bands, accounting for roughly 120 chromosomes, ranging in size from 50 kb up to the size of the largest, 5.7 Mb chromosome of Schizosaccharomyces pombe. Three different VSG gene expression sites were located to chromosomes of 430 kb, 1.5 Mb and 3 Mb, respectively. We have not been able to identify additional, previously cryptic DNA rearrangements, that could explain the activation or inactivation of the expression sites.