Cloning and characterization of the <i>Drosophila </i>homolog of the xeroderma pigmentosum complementation-group B correcting gene, <i>ERCC3</i>

Previously the human nucleotide excision repair gene ERCC3 was shown to be responsible for a rare combination of the autosomal recessive DNA repair disorders xeroderma pigmentosum (complementation group B) and Cockayne's syndrome (complementation group C). The human and mouse ERCC3 proteins contain several sequence motifs suggesting that it is a nucleic acid or chromatin binding helicase. To study the significance of these domains and the overall evolutionary conservation of the gene, the homolog from Drosophila melanogaster was isolated by low stringency hybridizations using two flanking probes of the human ERCC3 cDNA. The flanking probe strategy selects for long stretches of nucleotide sequence homology, and avoids isolation of small regions with fortuitous homology. In situ hybridization localized the gene onto chromosome III 67E3/4, a region devoid of known D.melanogaster mutagen sensitive mutants. Northern blot analysis showed that the gene is continuously expressed in all stages of fly development. A slight increase (2-3 times) of ERCC3Dm transcript was observed in the later stages. Two almost full length cDNAs were isolated, which have different 5′ untranslated regions (UTR). The SD4 cDNA harbours only one long open reading frame (ORF) coding for ERCC3Dm. Another clone (SD2), however, has the potential to encode two proteins: a 170 amino acids polypeptide starting at the optimal first ATG has no detectable homology with any other proteins currently in the data bases, and another ORF beginning at the suboptimal second startcodon which is identical to that of SD4. Comparison of the encoded ERCC3Dm protein with the homologous proteins of mouse and man shows a strong amino acid conservation (71% identity), especially in the postulated DNA binding region and seven 'helicase' domains. The ERCC3Dm sequence is fully consistent with the presumed functions and the high conservation of these regions strengthens their functional significance. Microinjection and DNA transfection of ERCC3Dm into human xeroderma pigmentosum (c.g. B) fibroblasts and group 3 rodent mutants did not yield detectable correction. One of the possibilities to explain these negative findings is that the D.melanogaster protein may be unable to function in a mammalian repair context.</p

Cloning and characterization of the <i>Drosophila </i>homolog of the xeroderma pigmentosum complementation-group B correcting gene, <i>ERCC3</i>

Previously the human nucleotide excision repair gene ERCC3 was shown to be responsible for a rare combination of the autosomal recessive DNA repair disorders xeroderma pigmentosum (complementation group B) and Cockayne's syndrome (complementation group C). The human and mouse ERCC3 proteins contain several sequence motifs suggesting that it is a nucleic acid or chromatin binding helicase. To study the significance of these domains and the overall evolutionary conservation of the gene, the homolog from Drosophila melanogaster was isolated by low stringency hybridizations using two flanking probes of the human ERCC3 cDNA. The flanking probe strategy selects for long stretches of nucleotide sequence homology, and avoids isolation of small regions with fortuitous homology. In situ hybridization localized the gene onto chromosome III 67E3/4, a region devoid of known D.melanogaster mutagen sensitive mutants. Northern blot analysis showed that the gene is continuously expressed in all stages of fly development. A slight increase (2-3 times) of ERCC3Dm transcript was observed in the later stages. Two almost full length cDNAs were isolated, which have different 5′ untranslated regions (UTR). The SD4 cDNA harbours only one long open reading frame (ORF) coding for ERCC3Dm. Another clone (SD2), however, has the potential to encode two proteins: a 170 amino acids polypeptide starting at the optimal first ATG has no detectable homology with any other proteins currently in the data bases, and another ORF beginning at the suboptimal second startcodon which is identical to that of SD4. Comparison of the encoded ERCC3Dm protein with the homologous proteins of mouse and man shows a strong amino acid conservation (71% identity), especially in the postulated DNA binding region and seven 'helicase' domains. The ERCC3Dm sequence is fully consistent with the presumed functions and the high conservation of these regions strengthens their functional significance. Microinjection and DNA transfection of ERCC3Dm into human xeroderma pigmentosum (c.g. B) fibroblasts and group 3 rodent mutants did not yield detectable correction. One of the possibilities to explain these negative findings is that the D.melanogaster protein may be unable to function in a mammalian repair context.</p

The Development of a Decision Tool for the Empiric Treatment of Suspected Urinary Tract Infection in Frail Older Adults: A Delphi Consensus Procedure

Objectives: Nonspecific signs and symptoms combined with positive urinalysis results frequently trigger antibiotic therapy in frail older adults. However, there is limited evidence about which signs and symptoms indicate urinary tract infection (UTI) in this population. We aimed to find consensus among an international expert panel on which signs and symptoms, commonly attributed to UTI, should and should not lead to antibiotic prescribing in frail older adults, and to integrate these findings into a decision tool for the empiric treatment of suspected UTI in this population. Design: A Delphi consensus procedure. Setting and Participants: An international panel of practitioners recognized as experts in the field of UTI in frail older patients. Measures: In 4 questionnaire rounds, the panel (1) evaluated the likelihood that individual signs and symptoms are caused by UTI, (2) indicated whether they would prescribe antibiotics empirically for combinations of signs and symptoms, and (3) provided feedback on a draft decision tool. Results: Experts agreed that the majority of nonspecific signs and symptoms should be evaluated for other causes instead of being attributed to UTI and that urinalysis should not influence treatment decisions unless both nitrite and leukocyte esterase are negative. These and other findings were incorporated into a decision tool for the empiric treatment for suspected UTI in frail older adults with and without an indwelling urinary catheter. Conclusions: A decision tool for suspected UTI in frail older adults was developed based on consensus among an international expert panel. Studies are needed to evaluate whether this decision tool is effective in reaching its aim: the improvement of diagnostic evaluation and treatment for suspected UTI in frail older adults