ADHD-associated dopamine transporter, latrophilin and neurofibromin share a dopamine-related locomotor signature in Drosophila.

Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neuropsychiatric disorder with hyperactivity as one of the hallmarks. Aberrant dopamine signaling is thought to be a major theme in ADHD, but how this relates to the vast majority of ADHD candidate genes is illusive. Here we report a Drosophila dopamine-related locomotor endophenotype that is shared by pan-neuronal knockdown of orthologs of the ADHD-associated genes Dopamine transporter (DAT1) and Latrophilin (LPHN3), and of a gene causing a monogenic disorder with frequent ADHD comorbidity: Neurofibromin (NF1). The locomotor signature was not found in control models and could be ameliorated by methylphenidate, validating its relevance to symptoms of the disorder. The Drosophila ADHD endophenotype can be further exploited in high throughput to characterize the growing number of candidate genes. It represents an equally useful outcome measure for testing chemical compounds to define novel treatment options.

Four-year clinical evaluation of a self-adhesive luting agent for ceramic inlays.

OBJECTIVES: The aim of this randomized controlled clinical trial was to evaluate the 4-year clinical performance of a self-adhesive resin cement, RelyX Unicem (3M ESPE), used for cementation of ceramic inlays. In addition, the influence of selectively acid-etching enamel prior to luting on the clinical performance of the restorations was assessed. METHODS: Sixty-two IPS Empress 2 inlays/onlays were placed in 31 patients by two experienced clinicians. The restorations were luted with RelyX Unicem with (=experimental group: E) or without (=control group: NE) prior enamel etching with phosphoric acid. At baseline, 6 months, and 1, 2, and 4 years after placement, the restorations were assessed by two calibrated investigators using modified USPHS criteria. Ten selected samples of each group were investigated under SEM regarding morphological changes at the cement-inlay interface. RESULTS: The recall rate at 4 years was 97%. Two restorations (1 E, 1 NE) were lost, and one (E) had to be replaced due to inlay and tooth fracture resulting in a survival rate of 95%. No significant differences between the experimental and control group were noticed regarding all criteria (McNemar, p < 0.05). An obvious deterioration in marginal integrity was observed after 4 years as only 5% (E = 7%; NE = 3%) of the restorations exhibited an excellent marginal adaptation. In 90% of the restorations small, still clinically acceptable marginal deficiencies were observed. SEM of the luting gap showed an increased wear of the RelyX Unicem cement over the 4-year period. CONCLUSIONS: The self-adhesive luting cement RelyX Unicem can be recommended for bonding of ceramic inlays/onlays. Additional selective enamel etching does not improve the clinical performance of the restorations within the 4-year period. CLINICAL RELEVANCE: The self-adhesive resin composite RelyX Unicem showed acceptable clinical performance after 4 years of clinical service.

Progress in Arabidopsis genome sequencing and functional genomics.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

PCR amplification and sequence of the p33 piroplasm surface antigen gene of a Theileria species isolated from cattle in west Java, Indonesia.

Oriental theileriosis, a parasitic disease of cattle caused by protozoa of the Theileria orientalis/sergenti/buffeli group, has been reported in Indonesia but its causal agent had not yet been characterized. This study was carried out to isolate and characterize the parasite through comparison of its p33 piroplasm surface antigen gene sequence, with known p32 sequences of T. sergenti and T. buffeli isolates. A Theileria spp. isolate was collected from an Ongole cow in Jonggol, West-Java, and transferred into a splenectomized calf for antigen production. Piroplasms were extracted from erythrocytes by ammonium chloride-lysis, separated from unlysed leukocytes and parasitic DNA was phenol-extracted. Polymerase chain reaction (PCR) was carried out on genomic DNA with a pair of 20 bp primers showing consensus for the p32-35 nucleotide sequence of 7 known T. orientalis/sergenti/buffeli isolates. An 875 bp fragment was amplified, and further sequenced on both strands by the dye-labeled terminators method. It showed an 88% homology with the p33 nucleotide sequence of the Japanese T. sergenti Ikeda stock and a lesser homology with 6 other sequences of Australian T. buffeli or Japanese T. sergenti stocks. It was shown to share the presence of the Pst 1 and the absence of the HindIII restriction sites of the T. sergenti Ikeda stock and of one Australian T. buffeli stock, respectively. In conclusion, the affiliation to and the relative position of this Indonesian isolate within the T. orientalis/sergenti/buffeli group has been elucidated.

Sequence analysis of a near-subtelomeric 35.4 kb DNA segment on the right arm of chromosome VII from Saccharomyces cerevisiae carrying the MAL1 locus reveals 15 complete open reading frames, including ZUO1, BGL2 and BIO2 genes and an ABC transporter gene.

The nucleotide sequence of 35,400 bp at approximately 10 kb from the right telomere of chromosome VII was determined. The segment contains the MAL1 locus, one of the five unlinked loci sufficient for maltose utilization. Until now, each of these loci was considered to contain three genes (for regulator, permease and alpha-glucosidase), but a fourth gene, presumably an extra alpha-glucosidase gene, was found at MAL1 adjacent to the usual cluster of three genes. The two glucosidase genes are present in opposite orientation, forming an inverted repeat structure. In addition to the four genes at MAL1, there are 11 complete, non-overlapping open reading frames (ORFs) longer than 300 bp in the sequence presented here. A new ABC transporter gene (YGR281w), required for oligomycin resistance was found (YOR1; Katzman et al., 1995), and the previously sequenced BGL2 (YGR282c), ZUO1 (YGR285c) and BIO2 (YGR286c) genes were located. The sequence of BIO2, a biotin synthetase gene, required substantial correction and the size of Bio2p is 375, rather than 356, amino acids. Two ORFs show rather weak similarities to animal genes: YGR278w to an unknown ORF of Caenorhabditis elegans and YGR284c to the murine Surf-4, a member of a cluster of at least four housekeeping genes. The remaining five ORFs do not encode known functions, but three of these show weak to high similarities to other ORFs in the Saccharomyces cerevisiae genome and one (YGR280c) codes for a particularly lysine-rich protein.

The sequence of a nearly unclonable 22.8 kb segment on the left arm chromosome VII from Saccharomyces cerevisiae reveals ARO2, RPL9A, TIP1, MRF1 genes and six new open reading frames.

The nucleotide sequence of 22,803 bp on the left arm of chromosome VII was determined by polymerase chain reaction-based approaches to compensate for the unstable character of cosmid clones from this region of the chromosome. The coding density of the sequence is particularly high (more than 83%). Twelve open reading frames (ORFs) longer than 300 bp were found, two of which (at the left side) have been described previously (James et al., 1995) after sequencing of an overlapping cosmid. Four other ORFs correspond to published sequences of the known genes ARO2, RPL9A, TIP1 and MRF1. ARO2 codes for chorismate synthetase. RPL9A for protein L9 of the large ribosomal subunit and MRF1 for a mitochondrial translation release factor. The TIP1 product interacts with Sec20p and is thus involved in transport from endoplasmic reticulum to Golgi. Five of the remaining ORFs have not been identified previously, while the sixth (YGL142c) has been partially sequenced as it lies 5' upstream of MRF1. These six ORFs are relatively large (between 933 and 3657 nucleotides). YGL146c, YGL142c, YGL140c and YGL139w have no significant homology to any protein sequence presently available in the public databases, but show two, nine, nine and eight putative transmembrane spans, respectively. YGL144c has a serine active site signature of lipases. YGL141w has limited homology to several human proteins, one of which mediates complex formation between papillomavirus E6 oncoprotein and tumor suppressor protein p53.

The sequence of 23 kb surrounding the SNF3 locus on the left arm of yeast chromosome IV reveals the location of five known genes and characterizes at least six new open reading frames including putative genes for ribosomal protein L35 and a sugar transport protein.

The nucleotide sequence of 22,846 bp of the left arm of chromosome IV is described. Twelve open reading frames (ORFs) greater than 100 triplets were detected, one of which extends into an adjacent cosmid. Two of the ORFs may contain an intron. One of these is an L35 ribosomal protein gene. Five ORFs (D1204, D1214, D1219, D1234 and D1244) encode previously sequenced genes (MGT1, SHM1, ASF2, SNF3 and ARF2, respectively). The nucleotide sequence of a sixth ORF (D1229) is quite similar to the WEB1 gene, which appeared in the DNA databases shortly after finishing the sequence reported here. It is not clear whether or not WEB1 and D1229 represent one and the same gene. The co-linearity of the reported DNA sequences with the genome of strains from Saccharomyces cerevisiae subspecies carlsbergensis, sake and diastaticus was assessed by comparative PCR with overlapping primer sets.

Expression of an ATP-binding cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae.

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

New open reading frames, one of which is similar to the nifV gene of Azotobacter vinelandii, found on a 12.5 kbp fragment of chromosome IV of Saccharomyces cerevisiae.

The nucleotide sequence of a 12.5 kbp segment of the left arm of chromosome IV is described. Five open reading frames (ORFs) longer than 100 amino acids were detected, all of which are completely confined to the 12.5 kbp region. Two ORFs (D1271 and D1286) correspond to previously sequenced genes (PPH22 and VMA1 or TFP1, respectively). ORF D1298 shows the characteristics of alpha-isopropylmalate and homocitrate synthase genes and is similar to the nifV gene of Azotobacter vinelandii. Two more ORFs have no apparent homologue in the data libraries. Conversely, two smaller ORFs of 25 and 85 amino acids encoding the ribosomal protein YL41A and an ATPase inhibitor, respectively, were detected. Although a substantial part of the 12.5 kbp fragment apparently lacks protein-coding characteristics, no other elements, such as tRNA genes or transposons, were found.

Twelve open reading frames revealed in the 23.6 kb segment flanking the centromere on the Saccharomyces cerevisiae chromosome XIV right arm.

The nucleotide sequence of 23.6 kb of the right arm of chromosome XIV is described, starting from the centromeric region. Both strands were sequenced with an average redundancy of 4.87 per base pair. The overall G+C content is 38.8% (42.5% for putative coding regions versus 29.4% for non-coding regions). Twelve open reading frames (ORFs) greater than 100 amino acids were detected. Codon frequencies of the twelve ORFs agree with codon usage in Saccharomyces cerevisiae and all show the characteristics of low level expressed genes. Five ORFs (N2019, N2029, N2031, N2048 and N2050) are encoded by previously sequenced genes (the mitochondrial citrate synthase gene, FUN34, RPC34, PRP2 and URK1, respectively). ORF N2052 shows the characteristics of a transmembrane protein. Other elements in this region are a tRNA(Pro) gene, a tRNA(Asn) gene, a tau 34 and a truncated delta 34 element. Nucleotide sequence comparison results in relocation of the SIS1 gene to the left arm of the chromosome as confirmed by colinearity analysis.

Nucleotide sequence analysis of an 8887 bp region of the left arm of yeast chromosome XIV, encompassing the centromere sequence.

The nucleotide sequencing of 8887 bp of the left arm of chromosome XIV is described. The sequence includes the centromeric region. Both strands were sequenced with an average redundancy of 5.09 per base pair. The overall G+C content is 37.3% (39.2% for putative coding regions versus 32.5% for non-coding regions). Six open reading frames (ORFs) greater than 100 amino acids were detected, all of which are completely confined to the 8.9 kbp region. Codon frequencies of the six ORFs agree with codon usage in Saccharomyces cerevisiae and all show the characteristics of low-level expressed genes. Comparison of the translated sequences with protein sequences in data bases suggests the presence of two ORFs (N2014 and N2007) encoding ribosomal proteins, the latter of which is the previously sequenced MRP7 gene. Another ORF (N2012) could encode a membrane-associated protein since it contains secretory signal sequence and two presumed transmembrane helices. This protein might be involved in mitochondrial energy transfer. ORF N2016 is immediately adjacent to the centromere, suggesting that it corresponds to the SPO1 gene, which is very tightly linked to the centromere at the left arm side of chromosome XIV (Mortimer et al., 1989).

A general method for routine sequencing of cloned DNA fragments using commercial dye primers and its application in sequence analysis of Toxoplasma gondii RH genome fragments.

A generally applicable approach for amplification and subsequent sequencing using commercially available dye primers is described. The polymerase chain reaction primers, one of which is biotinylated, are tagged at their 5' end with sequences of commercial sequencing primers. After purification and strand separation on streptavidin-coated magnetic beads, both strands are sequenced automatically on an Applied Biosystems 373A DNA sequencer using the appropriate standard dye-labeled sequencing primers. This approach is demonstrated on PstI and MnlI DNA fragments from Toxoplasma gondii RH cloned in the in-house developed derivatives of vector pJRtac99.

DNA sequencing by a subcloning-walking strategy using a specific and semi-random primer in the polymerase chain reaction.

In its basic concept, in vitro DNA amplification by the polymerase chain reaction (PCR) is restricted to those instances in which segments of known sequence flank the fragment to be amplified. Recently, techniques have been developed for amplification of unknown DNA sequences. These techniques, however, are dependent on the presence of suitable restriction endonuclease sites. Here, we describe a strategy for PCR amplification of DNA that lies outside the boundaries of known sequence. It is based on the use of one specific primer, homologous to the known sequence, and one semi-random primer. Restriction sites in the 5' proximal regions of both primers allow for cloning of the amplified DNA in a suitable sequencing vector or any other vector. It was shown by sequence analysis that the cloned DNA fragments represent contiguous DNA fragments that are flanked at one side by the sequence of the specific primer. When omitting the semi-random primer, a single clone was obtained, which originated from PCR amplification of target DNA by the specific primer in both directions.