Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila.

Proteins with expanded polyglutamine repeats cause Huntington's disease and other neurodegenerative diseases. Transcriptional dysregulation and loss of function of transcriptional co-activator proteins have been implicated in the pathogenesis of these diseases. Huntington's disease is caused by expansion of a repeated sequence of the amino acid glutamine in the abnormal protein huntingtin (Htt). Here we show that the polyglutamine-containing domain of Htt, Htt exon 1 protein (Httex1p), directly binds the acetyltransferase domains of two distinct proteins: CREB-binding protein (CBP) and p300/CBP-associated factor (P/CAF). In cell-free assays, Httex1p also inhibits the acetyltransferase activity of at least three enzymes: p300, P/CAF and CBP. Expression of Httex1p in cultured cells reduces the level of the acetylated histones H3 and H4, and this reduction can be reversed by administering inhibitors of histone deacetylase (HDAC). In vivo, HDAC inhibitors arrest ongoing progressive neuronal degeneration induced by polyglutamine repeat expansion, and they reduce lethality in two Drosophila models of polyglutamine disease. These findings raise the possibility that therapy with HDAC inhibitors may slow or prevent the progressive neurodegeneration seen in Huntington's disease and other polyglutamine-repeat diseases, even after the onset of symptoms.

Antisense-mediated down-regulation of the human huntingtin gene.

The present study determines whether the expression of the huntingtin gene might be subject to antisense (AS)-mediated down-regulation. A series of AS oligodeoxynucleotides (ODNs) complementary to the huntingtin transcript [i.e., nucleotide (nt) -25 to 35] were designed and synthesized, and the AS efficacy was investigated by using a combination of in vitro transcription and translation to mimic in vivo conditions. An oligomer directed to nt -1 to 15 (ODN III) markedly reduced the incorporation of [(3)H]leucine into the huntingtin gene product in a dose-dependent manner (ED(50) of approximately 11.5 microM). ODNs that overlap with ODN III on both 5'- and 3'-flanking regions also produced translation arrest of the huntingtin protein; however, the AS-mediated effect of these ODNs represented approximately 50% of the effect of ODN III. In contrast, an ODN directed to nt 19 to 35 had no AS effect. The efficacy of ODN III also was investigated in an inducible, stably transfected PC-12 cell line expressing a truncated huntingtin exon 1 protein. In accordance with the cell free translation studies, ODN III (1-10 microM) markedly decreased the abundance of the huntingtin-green fluorescence fusion protein to 40 to 46% of the control levels. In summary, a series of putative AS candidates were screened for down-regulation of the huntingtin gene, and an ODN molecule directed to the methionine initiation codon was identified with maximum AS effects.

Population genetics with RAPD-PCR markers: the breeding structure of Aedes aegypti in Puerto Rico.

RAPD-PCR polymorphisms at 57 presumptive loci were used to examine the breeding structure of the mosquito Aedes aegypti in Puerto Rico. Mosquitoes were sampled from 16 locations in six cities and samples were located in a nested spatial design to examine local patterns of gene flow. Allele frequencies were estimated assuming (1) that genomic regions amplified by RAPD-PCR segregate as dominant alleles, (2) that genotypes at RAPD loci are in Hardy-Weinberg proportions, (3) identity in state (iis) among dominant amplified alleles and (4) iis among null alleles. The average genic heterozygosity was 0.354, more than twice the level detected in earlier allozyme surveys. Nested analysis of variance indicated extensive genetic differentiation among locations within cities. Effective migration rates (Nm) among cities were estimated from FST assuming an island model of migration. Estimates of Nm ranged from 9.7 to 12.2 indicating a high dispersal rate. The large number of polymorphisms revealed by RAPD-PCR allowed the distribution of FST and linkage disequilibrium to be examined among loci and demonstrated that small samples inflate FST and linkage disequilibrium. No linkage disequilibrium maintained through epistasis was detected among alleles at the 57 loci.

Regional assignment and tissue expression of twenty-three expressed sequence tags (ESTs) from human chromosome 5.

Regional localization and expression patterns are reported for 19 expressed sequence tags (ESTs) from human chromosome 5, two of which were derived from the same transcript. Two of the ESTs correspond to genes not previously characterized in humans: a stress-activated protein kinase and nicotinamide nucleotide transhydrogenase. Expression was determined by three methods: Northern blots, PCR from tissue-specific cDNA libraries, and sequence sampling from EST sequencing projects. Six of the ESTs show no expression, and EST01986 appears to be expressed predominantly in the brain by all methods tested.

Use of randomly amplified polymorphic DNA amplified by polymerase chain reaction markers to estimate the number of Aedes aegypti families at oviposition sites in San Juan, Puerto Rico.

We report the application of a molecular genetic technique to estimate the number of full-sibling families of Aedes aegypti contained in oviposition traps. Randomly amplified polymorphic DNA amplified by the polymerase chain reaction markers were used to estimate the numbers and sizes of families in traps at field locations in San Juan, Puerto Rico. Forty-nine presumptive loci were amplified with five primers in a total of 813 individuals from 26 sites. The average family size was 10.95, but the size distribution was skewed with an excess of small families containing 1-2 individuals. The number of families increased with the number of eggs in traps; however, the average family size decreased as the number of eggs increased. This suggests that females oviposited only a few eggs in traps that were recently placed in the field and lacked mosquito eggs or fewer eggs were oviposited as traps became crowded.

Novel activity of a yeast ligase deletion polypeptide. Evidence for GTP-dependent tRNA splicing.

Yeast tRNA ligase possesses multiple activities which are required for the joining of tRNA halves during the tRNA splicing process: cyclic phosphodiesterase, kinase, adenylylate synthetase, and ligase. A deletion polypeptide of a dihydrofolate reductase-ligase fusion protein, designated DAC, was previously shown to join tRNA halves although ATP-dependent kinase activity was not measurable in the assay used. We describe here a characterization of the mechanism of joining used by DAC and the structure of the tRNA product. DAC produces a joined tRNA and a splice junction with a structure identical to that produced by DAKC, the full-length dihydrofolate reductase-ligase fusion. Furthermore, DAC can use GTP as the sole cofactor in the joining reaction, in contrast to DAKC, which can only complete splicing in the presence of ATP. Both enzymes exhibit GTP-dependent kinase activity at 100-fold greater efficiency than with ATP. These results suggest that a potential function for the center domain of tRNA ligase (missing in DAC) is to provide structural integrity and aid in substrate interactions and specificity. They also support the hypothesis that ligase may prefer to use two different cofactors during tRNA splicing.

Estimation of the number of full sibling families at an oviposition site using RAPD-PCR markers: applications to the mosquito Aedes aegypti.

There are many species in which groups of individuals encountered in the field are known to consist of mixtures of full-sibling families. We describe a statistical technique, based on the use of random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers, that allows for the estimation of the number of families contained in these groups. We test the technique on full-sibling families of the mosquito Aedes aegypti, a species that distributes its eggs among several locations. Mixtures of 10 families with 15 individuals per family were analyzed using 40 RAPD-PCR loci amplified by 5 primers. Our analysis accurately estimated the number of families. The technique was accurate when the number of families was small or when family sizes were small and variable.

Deletion analysis of a multifunctional yeast tRNA ligase polypeptide. Identification of essential and dispensable functional domains.

Splicing of tRNA precursors in extracts of Saccharomyces cerevisiae requires the action of two enzymes: a site specific endonuclease and a tRNA ligase. The tRNA ligase contains three distinct enzymatic activities: a polynucleotide kinase, a cyclic phosphodiesterase, and an RNA ligase. The polypeptide also has a high affinity pre-tRNA binding site based on its ability to form stable complexes with pre-tRNA substrates. To investigate the organization of functional enzymatic and binding elements within the polypeptide a series of defined tRNA ligase gene deletions were constructed and corresponding proteins were expressed in Escherichia coli as fusions with bacterial dihydrofolate reductase (DHFR). The DHFR/ligase derivative proteins were then efficiently purified by affinity chromatography. The complete ligase fusion protein retained enzymatic and binding activities which were unaffected by the presence of the DHFR segment. Examination of tRNA ligase deletion derivatives revealed that the amino-terminal region was required for adenylylation, while the carboxyl-terminal region was sufficient for cyclic phosphodiesterase activity. Deletions within the central region affected kinase activity. Pre-tRNA binding activity was not strictly correlated with a distinct enzymatic domain. A DHFR/ligase-derived protein lacking kinase activity efficiently joined tRNA halves. We postulate that this variant utilizes a novel RNA ligation mechanism.

Preferential binding of yeast tRNA ligase to pre-tRNA substrates.

Joining of tRNA halves during splicing in extracts of Saccharomyces cerevisiae requires each of the three enzymatic activities associated with the tRNA ligase polypeptide. Joining is most efficient for tRNA as opposed to oligonucleotide substrates and is sensitive to single base changes at a distance from splice sites suggesting considerable specificity. To examine the basis for this specificity, binding of ligase to labeled RNA substrates was measured by native gel electrophoresis. Ligase bound tRNA halves with an association constant 1600-fold greater than that for a nonspecific RNA. Comparison of binding of a series of tRNA processing intermediates revealed that tRNA-structure, particularly in the region around the splice sites, contributes to specific binding. Finally, the ligase was shown to form multiple, discrete complexes with tRNA substrates. The basis for recognition by ligase and its role in a tRNA processing pathway are discussed.