We produced a transgenic mosquito expressing a rodent malaria vaccine candidate antigen in the salivary gland. Three tandemly repeated amino acid units from the repeat region of circumsporozoite protein of Plasmodium berghei (PbCS3R) fused to red fluorescent protein (monomeric DsRed) was chosen as a vaccine candidate antigen. Immunoblot and fluorescence microscopic analyses showed the transgene expression in the female salivary gland. The transgene product was released from the proboscis as a component of saliva. The monomeric DsRed-fusion expression system could be suitable for transgene secretion in the saliva of female mosquitoes. Mice repeatedly bitten by transgenic mosquitoes raised antibodies against P. berghei sporozoites, and the sera had protective ability against sporozoite invasion of human hepatoma HepG2 cells. These results suggest that transgene products are immunogenically active in saliva, and induce the antibodies to malaria parasite. These findings indicate that this technology has the potential for production of a 'flying vaccinator' for rodent malaria parasites.

A tandem repeat in DNA is two or more contiguous, approximate copies of a pattern of nucleotides. Tandem repeats have been shown to cause human disease, may play a variety of regulatory and evolutionary roles and are important laboratory and analytic tools. Extensive knowledge about pattern size, copy number, mutational history, etc. for tandem repeats has been limited by the inability to easily detect them in genomic sequence data. In this paper, we present a new algorithm for finding tandem repeats which works without the need to specify either the pattern or pattern size. We model tandem repeats by percent identity and frequency of indels between adjacent pattern copies and use statistically based recognition criteria. We demonstrate the algorithm's speed and its ability to detect tandem repeats that have undergone extensive mutational change by analyzing four sequences: the human frataxin gene, the human beta T cellreceptor locus sequence and two yeast chromosomes. These sequences range in size from 3 kb up to 700 kb. A World Wide Web server interface atc3.biomath.mssm.edu/trf.html has been established for automated use of the program.

Added by telomerase, arrays of TTAGGG repeats specify the ends of human chromosomes. A complex formed by six telomere-specific proteins associates with this sequence and protects chromosome ends. By analogy to other chromosomal protein complexes such as condensin and cohesin, I will refer to this complex as shelterin. Three shelterin subunits, TRF1, TRF2, and POT1 directly recognize TTAGGG repeats. They are interconnected by three additional shelterin proteins, TIN2, TPP1, and Rap1, forming a complex that allows cells to distinguish telomeres from sites of DNA damage. Without the protective activity of shelterin, telomeres are no longer hidden from the DNA damage surveillance and chromosome ends are inappropriately processed by DNA repair pathways. How does shelterin avert these events? The current data argue that shelterin is not a static structural component of the telomere. Instead, shelterin is emerging as a protein complex with DNA remodeling activity that acts together with several associated DNA repair factors to change the structure of the telomeric DNA, thereby protecting chromosome ends. Six shelterin subunits: TRF1, TRF2, TIN2, Rap1, TPP1, and POT1.

Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human malignancy. An internal tandem duplication (ITD) of the juxtamembrane (JM) domain-coding sequence of the FLT3 gene (FLT3/ITD) is found in 20% of patients with acute myeloid leukemia (AML) and is strongly associated with leukocytosis and a poor prognosis. On the other hand, mutations of the c-KIT gene, which have been found in mast cell leukemia and AML, are clustered in 2 distinct regions, the JM domain and D816 within the activation loop. This study was designed to analyze the mutation of D835 of FLT3, which corresponds to D816 of c-KIT, in a large series of human hematologic malignancies. Several kinds of missense mutations were found in 30 of the 429 (7.0%) AML cases, 1 of the 29 (3.4%) myelodysplastic syndrome (MDS) cases, and 1 of the 36 (2.8%) acute lymphocytic leukemia patients. The D835Y mutation was most frequently found (22 of the 32 D835 mutations), followed by the D835V (5), and D835H (1), D835E (1), and D835N (1) mutations. Of note is that D835 mutations occurred independently of FLT3/ITD. An analysis in the 201 patients newly diagnosed with AML (excluding M3) revealed that, in contrast to the FLT3/ITD mutation (n = 46), D835 mutations (n = 8) were not significantly related to the leukocytosis, but tended to worsen disease-free survival. All D835-mutant FLT3 were constitutively tyrosine-phosphorylated and transformed 32D cells, suggesting these mutations were constitutively active. These results demonstrate that the FLT3 gene is the target most frequently mutated to become constitutively active in AML.

Constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplication (ITD) of the juxtamembrane region or by point mutations in the second tyrosine kinase domain (TKD), has been described in patients with acute myelogenous leukemia (AML). We analyzed the prevalence and the potential prognostic impact of FLT3 mutations in 979 AML patients. Results were correlated with cytogenetic data and the clinical response. FLT3-ITD mutations were found in 20.4% and FLT3-TKD mutations in 7.7% of the patients. Each mutation was associated with similar clinical characteristics and was more prevalent in patients with normal karyotype. Significantly more FLT3 aberrations were found in patients with FAB M5, and fewer were found in patients with FAB M2 and M6. Although less frequent in patients with cytogenetic aberrations, FLT3-ITDs were found in 13 of 42 patients with t(15;17) and in 9 of 10 patients with t(6;9). The prevalence of the ITD allele on the DNA level was heterogeneous, ranging from faint mutant bands in some patients to predominant mutant bands in others. Based on quantitative analysis, the mutant-wild-type (wt) ratio ranged from 0.03 to 32.56 (median, 0.78). Patients with a high mutant/wt ratio (ie, greater than 0.78) had significantly shorter overall and disease-free survival, whereas survival in patients with ratios below 0.78 did not differ from those without FLT3 aberrations. Multivariate analysis confirmed a high mutant/wt ratio to be a strong independent prognostic factor. Taken together, these data confirm that FLT mutations represent a common alteration in adult AML. Constitutive activation may be associated with monocytoid differentiation. A high mutant/wt ratio in ITD-positive patients appears to have a major impact on the prognostic relevance.

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes >/=300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.

FLT3 (fms-related tyrosine kinase/Flk2/Stk-2) is a receptor tyrosine kinase (RTK) primarily expressed on hematopoietic cells. In blasts from acute myelogenous leukemia (AML) patients, 2 classes of FLT3 activating mutations have been identified: internal tandem duplication (ITD) mutations in the juxtamembrane domain (25%-30% of patients) and point mutations in the kinase domain activation loop (7%-8% of patients). FLT3-ITD mutations are the most common molecular defect identified in AML and have been shown to be an independent prognostic factor for decreased survival. FLT3-ITD is therefore an attractive molecular target for therapy. SU11248 is a recently described selective inhibitor with selectivity for split kinase domain RTKs, including platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and KIT. We show that SU11248 also has potent activity against wild-type FLT3 (FLT3-WT), FLT3-ITD, and FLT3 activation loop (FLT3-Asp835) mutants in phosphorylation assays. SU11248 inhibits FLT3-driven phosphorylation and induces apoptosis in vitro. In addition, SU11248 inhibits FLT3-induced VEGF production. The in vivo efficacy of SU11248 was investigated in 2 FLT3-ITD models: a subcutaneous tumor xenograft model and a bone marrow engraftment model. We show that SU11248 (20 mg/kg/d) dramatically regresses FLT3-ITD tumors in the subcutaneous tumor xenograft model and prolongs survival in the bone marrow engraftment model. Pharmacokinetic and pharmacodynamic analysis in subcutaneous tumors showed that a single administration of an efficacious drug dose potently inhibits FLT3-ITD phosphorylation for up to 16 hours following a single dose. These results suggest that further exploration of SU11248 activity in AML patients is warranted.

BACKGROUND The injection of double-stranded RNA (dsRNA) has been shown to induce a potent sequence-specific inhibition of gene function in diverse invertebrate and vertebrate species. The homology-dependent posttranscriptional gene silencing (PTGS) caused by the introduction of transgenes in plants may be mediated by dsRNA. The analysis of Caenorhabditis elegans mutants impaired with dsRNA-mediated silencing and studies in plants implicate a biological role of dsRNA-mediated silencing as a transposon-repression and antiviral mechanism. RESULTS We investigated the silencing of testis-expressed Stellate genes by paralogous Su(Ste) tandem repeats, which are known to be involved in the maintenance of male fertility in Drosophila melanogaster. We found that both strands of repressor Su(Ste) repeats are transcribed, producing sense and antisense RNA. The Stellate silencing is associated with the presence of short Su(Ste) RNAs. Cotransfection experiments revealed that Su(Ste) dsRNA can target and eliminate Stellate transcripts in Drosophila cell culture. The short fragment of Stellate gene that is homologous to Su(Ste) was shown to be sufficient to confer Su(Ste)-dependent silencing of a reporter construct in testes. We demonstrated that Su(Ste) dsRNA-mediated silencing affects not only Stellate expression but also the level of sense Su(Ste) RNA providing a negative autogenous regulation of Su(Ste) expression. Mutation in the spindle-E gene relieving Stellate silencing also leads to a derepression of the other genomic tandem repeats and retrotransposons in the germline. CONCLUSIONS Homology-dependent gene silencing was shown to be used to inhibit Stellate gene expression in the D. melanogaster germline, ensuring male fertility. dsRNA-mediated silencing may provide a basis for negative autogenous control of gene expression. The related surveillance system is implicated to control expression of retrotransposons in the germline.

Systemic lupus erythematosus (SLE, OMIM 152700) is a complex autoimmune disease that affects 0.05% of the Western population, predominantly women. A number of susceptibility loci for SLE have been suggested in different populations, but the nature of the susceptibility genes and mutations is yet to be identified. We previously reported a susceptibility locus (SLEB2) for Nordic multi-case families. Within this locus, the programmed cell death 1 gene (PDCD1, also called PD-1) was considered the strongest candidate for association with the disease. Here, we analyzed 2,510 individuals, including members of five independent sets of families as well as unrelated individuals affected with SLE, for single-nucleotide polymorphisms (SNPs) that we identified in PDCD1. We show that one intronic SNP in PDCD1 is associated with development of SLE in Europeans (found in 12% of affected individuals versus 5% of controls; P = 0.00001, r.r.(relative risk)= 2.6) and Mexicans (found in 7% of affected individuals versus 2% of controls; P = 0.0009, r.r.= 3.5). The associated allele of this SNP alters a binding site for the runt-related transcription factor 1 (RUNX1, also called AML1) located in an intronic enhancer, suggesting a mechanism through which it can contribute to the development of SLE in humans.

Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.

The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms.