DNA twisting flexibility and the formation of sharply looped protein-DNA complexes.

Gene-regulatory complexes often require that pairs of DNA-bound proteins interact by looping-out short (often approximately 100-bp) stretches of DNA. The loops can vary in detailed length and sequence and, thus, in total helical twist, which radically alters their geometry. How this variability is accommodated structurally is not known. Here we show that the inherent twistability of 89- to 105-bp DNA circles exceeds theoretical expectation by up to 400-fold. These results can be explained only by greatly enhanced DNA flexibility, not by permanent bends. They invalidate the use of classic theories of flexibility for understanding sharp DNA looping but support predictions of two recent theories. Our findings imply an active role for DNA flexibility in loop formation and suggest that variability in the detailed helical twist of regulatory loops is accommodated naturally by the inherent twistability of the DNA.

Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages.

HK620 is a temperate lambdoid bacteriophage that adsorbs to the O-antigen of its host, Escherichia coli H. The genome of a temperature-sensitive clear-plaque mutant consists of 38,297 nucleotides in which we recognize 60 open reading frames (orfs). Eighteen of these lie in a region of the genome that we call the virion structure domain. The other 42 orfs lie in what we call the metabolic domain. Virions of HK620 resemble those of phage P22. The virion structural orfs encode three kinds of putative proteins relative to the virion proteins of P22: (1) those that are nearly (about 90 %) identical; (2) those that are weakly (about 30 %) identical; and (3) those composed of nearly and weakly identical segments. We hypothesize that these composite proteins form bridges between the virion proteins of the other two kinds. Three of the putative virion proteins that are only weakly identical to P22 proteins are 71, 60 and 79 % identical to proteins encoded by the phage APSE-1, whose virions also resemble those of P22. Because the hosts of APSE-1 and HK620 have been separated from each other by an estimated 200 My, we propose using the amino acid differences that have accumulated in these proteins to estimate a biological clock for temperate lambdoid phages. The putative transcriptional regulatory gene circuitry of HK620 seems to resemble that of phage lambda. Integration, on the other hand, resembles that of satellite phage P4 in that the attP sequence lies between the leftward promoter and int rather than downstream of int. Comparing the metabolic domains of several lambdoid phage genomes reveals seven short conserved sequences roughly defining boundaries of functional modules. We propose that these boundary sequences are foci of genetic recombination that serve to assort the modules and make the metabolic domain highly mosaic genetically.

Kinetic trapping of DNA by transcription factor IIIB.

High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in yeast comprise the TATA-binding protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90. The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes. This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation. Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA. Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (DeltaG degrees = -12.10 +/- 0.12 kcal/mol) and TFIIIB-DNA (DeltaG degrees = -11.90 +/- 0.14 kcal/mol) complexes are equivalent whereas a kinetic analysis shows that the half-lives of these complexes (46 +/- 3 min and 95 +/- 6 min, respectively) differ significantly. The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes and increase the barrier to complex dissociation.

Comprehensive genome sequence analysis of a breast cancer amplicon.

Gene amplification occurs in most solid tumors and is associated with poor prognosis. Amplification of 20q13.2 is common to several tumor types including breast cancer. The 1 Mb of sequence spanning the 20q13.2 breast cancer amplicon is one of the most exhaustively studied segments of the human genome. These studies have included amplicon mapping by comparative genomic hybridization (CGH), fluorescent in-situ hybridization (FISH), array-CGH, quantitative microsatellite analysis (QUMA), and functional genomic studies. Together these studies revealed a complex amplicon structure suggesting the presence of at least two driver genes in some tumors. One of these, ZNF217, is capable of immortalizing human mammary epithelial cells (HMEC) when overexpressed. In addition, we now report the sequencing of this region in human and mouse, and on quantitative expression studies in tumors. Amplicon localization now is straightforward and the availability of human and mouse genomic sequence facilitates their functional analysis. However, comprehensive annotation of megabase-scale regions requires integration of vast amounts of information. We present a system for integrative analysis and demonstrate its utility on 1.2 Mb of sequence spanning the 20q13.2 breast cancer amplicon and 865 kb of syntenic murine sequence. We integrate tumor genome copy number measurements with exhaustive genome landscape mapping, showing that amplicon boundaries are associated with maxima in repetitive element density and a region of evolutionary instability. This integration of comprehensive sequence annotation, quantitative expression analysis, and tumor amplicon boundaries provide evidence for an additional driver gene prefoldin 4 (PFDN4), coregulated genes, conserved noncoding regions, and associate repetitive elements with regions of genomic instability at this locus.

PAX3 gene structure, alternative splicing and evolution.

PAX3 is a member of the paired box family of transcription factors that function during embryogenesis and cancer epigenesis. Mutations in PAX3 cause Waardenburg syndrome (types 1 and 3), Craniofacial-deafness-hand syndrome and alveolar rhabdomyosarcoma in humans and the Splotch phenotype in mice. In this study, we describe the genomic structure of PAX3, including novel coding sequences and the complete 3' UTR. Alternative transcripts of PAX3 were identified in various tissues, including human adult skeletal muscle and mouse embryos. One of the novel alternative transcripts is evolutionarily conserved in quail and can transactivate a reporter construct containing the mouse c-met promoter. The sequences and alternative transcripts reported herein extend our understanding of the function and evolution of PAX3 in vertebrates and enable a comprehensive mutation screen for individuals with Waardenburg syndrome.

Salt-induced hypertension in Dahl salt-resistant and salt-sensitive rats with NOS II inhibition.

Although recent evidence suggests that reduced nitric oxide (NO) production may be involved in salt-induced hypertension, the specific NO synthase (NOS) responsible for the conveyance of salt sensitivity remains unknown. To determine the role of inducible NOS (NOS II) in salt-induced hypertension, we treated Dahl salt-resistant (DR) rats with the selective NOS II inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) for 12 days. Tail-cuff systolic blood pressures rose 29 +/- 6 and 42 +/- 8 mmHg in DR rats given 150 and 300 nmol AMT/h, respectively (P < 0.01, 2-way ANOVA) after 7 days of 8% NaCl diet. We observed similar results with two other potent selective NOS II inhibitors, S-ethylisourea (EIT) and N-[3-(aminomethyl)benzyl]acetamidine hydrochloride (1400W). Additionally, AMT effects were independent of alterations in endothelial function as assessed by diameter change of mesenteric arterioles in response to methacholine using videomicroscopy. We, therefore, conclude from these data that NOS II is important in salt-induced hypertension.

Positional cloning of ZNF217 and NABC1: genes amplified at 20q13.2 and overexpressed in breast carcinoma.

We report here the molecular cloning of an approximately 1-Mb region of recurrent amplification at 20q13.2 in breast cancer and other tumors and the delineation of a 260-kb common region of amplification. Analysis of the 1-Mb region produced evidence for five genes, ZNF217, ZNF218, and NABC1, PIC1L (PIC1-like), CYP24, and a pseudogene CRP (Cyclophillin Related Pseudogene). ZNF217 and NABC1 emerged as strong candidate oncogenes and were characterized in detail. NABC1 is predicted to encode a 585-aa protein of unknown function and is overexpressed in most but not all breast cancer cell lines in which it was amplified. ZNF217 is centrally located in the 260-kb common region of amplification, transcribed in multiple normal tissues, and overexpressed in all cell lines and tumors in which it is amplified and in two in which it is not. ZNF217 is predicted to encode alternately spliced, Kruppel-like transcription factors of 1,062 and 1,108 aa, each having a DNA-binding domain (eight C2H2 zinc fingers) and a proline-rich transcription activation domain.

Airway surface liquid thickness as a function of lung volume in small airways of the guinea pig.

The average thickness and distribution of airway surface liquid (ASL) on the luminal surface of peripheral airways were measured in normal guinea pig lungs frozen at functional residual capacity (FRC) and total lung capacity (TLC). Tissue blocks containing cross sections of airways of internal perimeter 0.188-3.342 mm were cut from frozen lungs and imaged by low-temperature scanning electron microscopy (LTSEM). Measurements made from LTSEM images were found to be independent of freezing rate by comparison of measurements at rapid and slow freezing rates. At both lung volumes, the ASL was not uniformly distributed in either the circumferential or longitudinal direction; there were regions of ASL where its thickness was < 0.1 micron, whereas in other regions ASL collected in pools. Discernible liquid on the surfaces of airways frozen at FRC followed the contours of epithelial cells and collected in pockets formed by neighboring cells, a geometry consistent with a low value of surface tension at the air-liquid interface. At TLC airway liquid collected to cover epithelial cells and to form a liquid meniscus, a geometry consistent with a higher value of surface tension. The average ASL thickness (h) was approximately proportional to the square root of airway internal perimeter, regardless of lung volume. For airways of internal perimeter 250 and 1,800 microns, h was 0.9 and 1.8 microns at FRC and 1.7 and 3.7 microns at TLC, respectively. For a given airway internal perimeter, h was 1.99 times thicker at TLC than at FRC; the difference was statistically significant (P < 0.01; 95% confidence interval 1.29-3.08).(ABSTRACT TRUNCATED AT 250 WORDS)

cDNA structure and in situ localization of the Aplysia californica pro-hormone convertase PC2.

The complete cDNA structure of the Aplysia californica pro-protein and pro-hormone convertase PC2 (aPC2) was obtained from a cDNA library of the nervous system. The deduced amino acid sequence revealed that aPC2 exhibits an 85%, 61% and 62% sequence identity to the Lymnaea stagnalis, Xenopus laevis and mouse PC2 homologues, respectively. The deduced stagnalis, Xenopus laevis and mouse PC2 homologues, respectively. The deduced primary sequence suggested a protein of 653 amino acids which includes a 27- and 88-amino acid signal peptide and pro-segment. The signal peptide and the C-terminal segments are the least conserved regions. On Northern blots of nervous system we detected a transcript of 6.8 kb. The in situ hybridization histochemistry on the abdominal ganglion revealed intense labeling of the bag cells. Large peptidergic cells and clusters of sensory and motor neurons also contained high levels of aPC2 mRNA.

Ecogenetics of Parkinson's disease: 4-hydroxylation of debrisoquine.

It is postulated that Parkinson's disease is the result of environmental factors acting on genetically susceptible individuals against a background of normal ageing. Many potentially neurotoxic xenobiotics are detoxified by hepatic cytochrome P450. The function of one such system was studied in forty patients with Parkinson's disease and forty normal control subjects. Significantly more parkinsonian than control subjects had partially or totally defective 4-hydroxylation of debrisoquine. Poor metabolisers of debrisoquine tended to have had earlier onset of disease.

Lysosomal enzymes in ataxia: discovery of two new cases of late onset hexosaminidase A and B deficiency (adult Sandhoff disease) in French Canadians.

We have measured in leukocytes the following lysosomal enzymes in 11 Friedreich disease cases, 11 "atypical" recessive ataxias, 13 neurological controls and 16 normal controls: hexosaminidase A and B; beta-galactosidase and neuraminidase (labile and cold stable, or A and B). The lysosomal enzyme deficiencies known to produce certain forms of spinocerebellar degeneration were not present in Friedreich's disease or the Charlevoix-Saguenay syndrome. The very small scale survey of "atypical" recessive ataxias revealed 3 cases of severe deficiencies in hexosaminidase activity. Two adult brothers presenting with the clinical phenotype of Kugelberg-Welander disease (one also with ataxia), were shown to have a severe deficiency of both HEX A and HEX B activity (Sandhoff biochemical pattern). This is the first such report. A further adult female patient, unrelated to the others, had a severe isolated deficiency of HEX B and presented with a very slowly progressive and mild ataxia with severe internal strabismus. These patients and their families are being studied clinically and biochemically in greater detail and will be reported elsewhere. However these preliminary findings justify screening for such lysosomal defects in all cases of "atypical" recessive ataxia.

Cardiac malic enzyme in Friedreich's disease.

We measured the activity of cytosolic and of mitochondrial malic enzyme in the hearts from 4 patients with Friedreich's disease and from two non-ataxic control subjects. There was a wide variability in the results and the slight overall decreases in both enzyme activities were not considered to be statistically significant. From these and other results, we conclude that deficient mitochondrial malic enzyme activity is not a constant or primary feature of Friedreich's disease.

A possible genetic pattern of taurine urinary excretion in Friedreich's Ataxia.

The taurine urinary excretion pattern, before and after an oral load of 250 mg taurine, was studied in normal control subjects and in patients with typical Friedreich's ataxia. It was demonstrated that in both situations the ataxic patients fell within the sub-types of "intermediate" and "high taurine excretors" while non were "low taurine excretors". It was also demonstrated that the excretion of taurine after a load in the obligate heterozygotes parents of the ataxic patients was intermediate between normal controls and patients. It is postulated that patients with Friedreich's Ataxia lack normal regulation of the high affinity-low capacity uptake system for taurine (the TH system) in the brush border of kidney tubules. The low affinity-high capacity uptake system in the same membranes (the TL system) appears to be normal in Friedreich's patients. The normal allele could be called THN and the variant THF and this trait would be inherited in an autosomal recessive fashion if it is linked to the Friedreich phenotype. Whether this finding is or is not the basic genetic defect in Friedreich's Ataxia will require more studies to clarify, but it is of interest to note that a similar pattern appears to be present in the fibroblasts of these patients.

Reduced formation of hippuric acid after oral benzoic acid in Friedreich's ataxia.

We have observed a markedly decreased formation of hippuric acid after benzoic acid load in patients with typical Friedreich's Ataxia compared to normal control subjects. Since there is evidence for normal or even enhanced tauro-conjugation in the bile of patients with this disease, with a decreased G/T ratio, it is unlikely that co-factor or enzyme concentrations are the cause of this defect. We postulate decreased availability of the enzyme for glycine conjugation either to bile acids in the usual situation or to benzoic acid in the artefactual test condition. This could be due to the enzyme's preference for an increased amount of taurine substrate in the liver. The relationship of this observation to the other biochemical changes observed in Friedreich's Ataxia must still be established.

Leukocyte valine dehydrogenase activity in Friedreich's ataxia.

We studied the activity of valine dehydrogenase (VDH) in leukocytes of 14 Friedreich's ataxia patients and of 14 normal control subjects. There was a significant 26% mean decrease in enzyme activity in the patients, a finding which could be responsible for the chronic accumulation of some alpha-keto acids with toxic metabolic consequences in that disease. However the deficiency was not present in all patients with the typical symptoms, nor was its magnitude sufficient to be considered the primary genetic defect in Friedreich's Ataxia.

Leucocyte glutamate dehydrogenase in various hereditary ataxias.

Leucocyte Glutamate Dehydrogenase (GDH) activity was measured in 44 patients with various forms of ataxia and 44 age and sex-matched normal controls. The only significant change found was a moderate decrease in activity in Friedreich's ataxia and a few patients with OPCA. This decreased activity is not primary to the disease but probably reflects a regulatory defect affecting mitochondrial membranes in these patients.