Power-laws in interferon-B mRNA distribution in virus-infected dendritic cells.

Interferon-beta (IFNB1) mRNA shows very large cell-to-cell variability in primary human dendritic cells infected by Newcastle disease virus, with copy numbers varying from a few to several thousands. Analysis of data from the direct measurement of the expression of this gene in its natural chromatin environment in primary human cells shows that the distribution of mRNA across cells follows a power law with an exponent close to -1, and thus encompasses a range of variation much more extensive than a Gaussian. We also investigate the single cell levels of IFNB1 mRNA induced by infection with Texas influenza A mutant viruses, which vary in their capacity to inhibit the signaling pathways responsible for activation of this gene. Here as well we observe power-law behavior for the distribution of IFNB1 mRNA, albeit over a truncated range of values, with exponents similar to the one for cells infected by Newcastle disease virus. We propose a model of stochastic enhanceosome and preinitiation complex formation that incorporates transcriptional pulsing. Analytical and numerical results show good agreement with the observed power laws, and thus support the existence of transcriptional pulsing of an unmodified, intact gene regulated by a natural stimulus.

Structure and mechanism of the RuvB Holliday junction branch migration motor.

The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks. The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain. The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution. ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces. Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins. Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions.

The ruv proteins of Thermotoga maritima: branch migration and resolution of Holliday junctions.

In homologous recombination in bacteria, the RuvAB Holliday junction-specific helicase catalyzes Holliday junction branch migration, and the RuvC Holliday junction resolvase catalyzes formation of spliced or patched structures. RuvAB and RuvC from the hyperthermophile Thermotoga maritima were expressed in Escherichia coli and purified to homogeneity. An inverted repeat sequence with unique termini was produced by PCR, restriction endonuclease cleavage, and head-to-tail ligation. A second inverted repeat sequence was derived by amplification of a second template containing a three-nucleotide insertion. Reassociation products from a mixture of these two sequences were homoduplex linear molecules and heteroduplex heat-stable Holliday junctions, which acted as substrates for both T. maritima RuvAB and RuvC. The T. maritima RuvAB helicase catalyzed energy-dependent Holliday junction branch migration at 70 degrees C, leading to heteroduplex linear duplex molecules with two three-nucleotide loops. Either ATP or ATP gamma S hydrolysis served as the energy source. T. maritima RuvC resolved Holliday junctions at 70 degrees C. Remarkably, the cleavage site was identical to the preferred cleavage site for E. coli RuvC [(A/T)TT(downward arrow)(G/C)]. The conservation of function and the ease of purification of wild-type and mutant thermophilic proteins argues for the use of T. maritima proteins for additional biochemical and structural studies.

Pesticides and inner-city children: exposures, risks, and prevention.

Six million children live in poverty in America's inner cities. These children are at high risk of exposure to pesticides that are used extensively in urban schools, homes, and day-care centers for control of roaches, rats, and other vermin. The organophosphate insecticide chlorpyrifos and certain pyrethroids are the registered pesticides most heavily applied in cities. Illegal street pesticides are also in use, including tres pasitos (a carbamate), tiza china, and methyl parathion. In New York State in 1997, the heaviest use of pesticides in all counties statewide was in the urban boroughs of Manhattan and Brooklyn. Children are highly vulnerable to pesticides. Because of their play close to the ground, their hand-to-mouth behavior, and their unique dietary patterns, children absorb more pesticides from their environment than adults. The long persistence of semivolatile pesticides such as chlorpyrifos on rugs, furniture, stuffed toys, and other absorbent surfaces within closed apartments further enhances urban children's exposures. Compounding these risks of heavy exposures are children's decreased ability to detoxify and excrete pesticides and the rapid growth, development, and differentiation of their vital organ systems. These developmental immaturities create early windows of great vulnerability. Recent experimental data suggest, for example, that chlorpyrifos may be a developmental neurotoxicant and that exposure in utero may cause biochemical and functional aberrations in fetal neurons as well as deficits in the number of neurons. Certain pyrethroids exert hormonal activity that may alter early neurologic and reproductive development. Assays currently used for assessment of the toxicity of pesticides are insensitive and cannot accurately predict effects to children exposed in utero or in early postnatal life. Protection of American children, and particularly of inner-city children, against the developmental hazards of pesticides requires a comprehensive strategy that monitors patterns of pesticide use on a continuing basis, assesses children's actual exposures to pesticides, uses state-of-the-art developmental toxicity testing, and establishes societal targets for reduction of pesticide use.

Methanococcus jannaschii flap endonuclease: expression, purification, and substrate requirements.

The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity. FEN retained activity after preincubation at 95 degrees C+ for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5' end adjacent to the single-strand-duplex junction. Deletion of the free 3' end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3' end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5' end failed to block cleavage, although this substrate was refractory to cleavage by the 5'-3' exonuclease activity of Taq DNA polymerase. For verification, the free 5' end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for Taq DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5' single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion phiX174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.

Effect of the delta-aminolevulinate dehydratase polymorphism on the accumulation of lead in bone and blood in lead smelter workers.

Lead inhibition of the zinc metalloenzyme delta-aminolevulinate dehydratase (ALAD) is one of the most sensitive indicators of blood lead levels. ALAD is polymorphic, with about 20% of Caucasians expressing the rarer ALAD2 allele. Previous studies indicated that this polymorphism may be a genetic factor in lead transport, metabolism, and/or distribution. Whole blood lead, serum lead, and ALAD genotype were determined for 381 lead smelter workers, including 70 workers expressing the ALAD2 allele, whose blood lead elevations were observed for more than 20 years of employment. The same employees demonstrated higher serum lead levels. Using a cumulative blood lead index (CBLI) for each worker, based on individual blood lead histories, and in vivo X-ray fluorescence measurements of bone lead to estimate total lead body burden, the slopes of linear relations of bone lead to CBLI were greater for workers homoallelic for ALAD1, indicating more efficient uptake of lead from blood into bone. This effect was most significant in calcaneus bone and for workers hired since 1977 [ALAD1-1: 0.0528 +/- 0.0028 and ALAD1-2 or 2-2: 0.0355 +/- 0.0031 (P < 0.001)]. Decreased transfer of blood lead into bone in individuals expressing the ALAD2 allele contrasted with increased blood lead. Thus the ALAD genotype affected lead metabolism and potentially modified lead delivery to target organs including the brain; however, the ALAD genotype did not significantly affect the net accumulation of lead in bone.

Lead binding to delta-aminolevulinic acid dehydratase (ALAD) in human erythrocytes.

Over 99% of the lead present in blood is usually found in erythrocytes. To investigate the nature of this selective accumulation of lead in erythrocytes, the specific binding of lead to proteins in human erythrocytes was studied using liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS). The principal lead-binding protein had a mass of approximately 240 kDa, and adsorption to specific antibodies showed that protein was delta-aminolevulinic acid dehydratase (ALAD). Thus, the previous notion that lead in erythrocytes was bound primarily to haemoglobin has to be revised. Furthermore, in lead-exposed workers, the percentage of lead bound to ALAD was influenced by a common polymorphism in the ALAD gene. Specifically, in seven carriers of the ALAD2 allele, 84% of the protein-bound lead recovered was bound to ALAD compared to 81% in seven homozygotes for the ALAD1 allele whose erythrocytes were matched for blood-lead concentration. The small difference was statistically significant in Wilcoxon matched-pairs signed-rank test (P = 0.03). No ALAD allele-specific difference in ALAD-bound lead was found among 20 unexposed controls. Perhaps the difference in ALAD-bound lead can provide an explanation for the previously reported finding of higher blood-lead levels among carriers of the ALAD2 allele than among ALAD1 homozygotes in lead-exposed populations.

Delta-aminolevulinic acid dehydratase polymorphism: influence on lead levels and kidney function in humans.

Delta-aminolevulinic acid dehydratase (ALAD) polymorphism has been reported to modify lead pharmacokinetics (i.e., individuals who express the ALAD2 allele [ALAD2 subjects] have higher blood lead levels than homozygotes for the ALAD1 allele [ALAD1 subjects]). In our study of 89 lead-exposed workers (7 ALAD2 homozygotes or heterozygotes) and 34 unexposed workers (10 ALAD2 heterozygotes), concentrations of urinary calcium and creatinine were lower in ALAD2 subjects than in ALAD1 subjects (respective medians: calcium--78 mg/l versus 185 mg/l, p = .003; creatinine--11.2 mmol/l versus 14.9 mmol/l, p = .008). No association was found between ALAD genotype and blood lead levels or bone lead levels. However, expression of the ALAD2 allele occurred less frequently among lead-exposed workers than in unexposed controls. The results indicated the presence of ALAD allele-specific differences in kidney function, as well as a possible genetic healthy-worker selection.

A murine model of genetic susceptibility to lead bioaccumulation.

Previous reports have shown that blood lead levels in humans are associated with a polymorphic form of delta-aminolevulinate dehydratase (ALAD), an enzyme of heme biosynthesis that binds and is inhibited by lead. We hypothesized that ALAD levels may influence the distribution and accumulation of lead in the blood and target organs. To assess this, we studied strains of mice that differ in the numbers of copies of the ALAD gene. Our findings showed that mice with a duplication of the ALAD gene (DBA) accumulated twice the amount of lead in their blood and had higher lead levels in kidney and liver than mice with a single copy of the gene (C57) exposed to the same oral doses of lead during adulthood. Hybrid animals showed intermediate blood lead levels. Levels of blood zinc protoporphyrin (ZPP) increased with lead exposure in C57 animals while they were not affected in DBA mice, suggesting protection from production of this abnormal enzyme in mice with a duplication of the gene. Except for these protective effects in the formation of ZPP in DBA animals, duplication of the ALAD gene was found to increase lead accumulation. We conclude that although these mouse strains do not precisely replicate the polymorphism observed in humans, they may be used as a model to study genetic influences in lead bioaccumulation. Understanding genetic factors that affect susceptibility to lead-induced intoxication could have important implications for public health and intervention initiatives. These mouse strains may represent a useful model for future study of the role of ALAD in lead intoxication.

Cloning, sequencing, and expression of ruvB and characterization of RuvB proteins from two distantly related thermophilic eubacteria.

The ruvB genes of the highly divergent thermophilic eubacteria Thermus thermophilus and Thermotoga maritima were cloned, sequenced, and expressed in Escherichia coli. Both thermostable RuvB proteins were purified to homogeneity. Like E. coli RuvB protein, both purified thermostable RuvB proteins showed strong double-stranded DNA-dependent ATPase activity at their temperature optima (> or = 70 degrees C). In the absence of ATP, T. thermophilus RuvB protein bound to linear double-stranded DNA with a preference for the ends. Addition of ATP or gamma-S-ATP destabilized the T. thermophilus RuvB-DNA complexes. Both thermostable RuvB proteins displayed helicase activity on supercoiled DNA. Expression of thermostable T. thermophilus RuvB protein in the E. coli ruvB recG mutant strain N3395 partially complemented the UV-sensitive phenotype, suggesting that T. thermophilus RuvB protein has a function similar to that of E. coli RuvB in vivo.

Insert selection by BamHI methyltransferase protection in P1 phage-based cloning.

A P1-based cloning system has been tested which depends upon (i) in vitro selection for vectors containing inserts mediated by methyltransferase (MTase) protection, and (ii) in vivo vector arm Cre-mediated recombination following electroporation. Specifically, chromosomal DNA was digested with BglII, dephosphorylated, methylated with BamHI MTase and ligated into the BamHI site of the vector, thereby destroying that site. Subsequent BamHI digestion acted as the in vitro selection, eliminating vector religation products prior to electroporation into cells expressing the Cre recombinase. Electroporation with linearized vector gave approx. 10(6) transformants per microgram vector, depending on vector concentration. Cloning of BglII fragments of gamma DNA using the in vitro selection system led to 1.3-4-fold fewer transformants per microgram vector. Plasmids recovered from these clones were all found to contain a gamma BglII fragment and the representation of fragments in the clones was independent of the length of the fragments. Both in vitro selection and electroporation are applicable to library construction using size-selected human DNA, with size selection either before or after ligation and BamHi digestion.

Unraveling the chronic toxicity of lead: an essential priority for environmental health.

Although population exposure to lead has declined, chronic lead toxicity remains a major public health problem in the United States affecting millions of children and adults. Important gaps exist in knowledge of the pathophysiology of chronic lead intoxication. These gaps have impeded development of control strategies. To close current gaps in knowledge of chronic lead toxicity, we propose an integrated, multidisciplinary, marker-based research program. This program combines a) direct measurement of individual lead burden by 109Cd X-ray fluorescence analysis of lead in bone, b) determination of ALA-D phenotype, an index of individual susceptibility to lead, and c) assessments of subclinical injury produced by lead in the kidneys, nervous system and, reproductive organs. Data from this research will provide answers to questions of great public health importance: a) Are current environmental and occupational standards adequate to prevent chronic lead intoxication? b) is lead mobilized from the skeleton during pregnancy or lactation to cause fetal toxicity? c) Is lead mobilized from bone during menopause to cause neurotoxicity? d) What is the significance of genetic variation in determining susceptibility to lead? e) What is the contribution of lead to hypertension, renal disease, chronic neurodegenerative disease or declining sperm counts? f) Is chelation therapy effective in reducing body lead burden in persons with chronic overexposure to lead?

Some class-IIS restriction endonucleases can cleave across a three-way DNA junction.

Three-way DNA junctions were synthesized with class-IIS restriction endonuclease (ENase) recognition sites and potential cleavage sites located on separate arms. Cleavage was investigated with junctions labeled in each of the three strands. BpmI and BsaI failed to cleave either strand of either arm, whereas BsmAI cleaved one strand. FokI and HphI cleaved both strands of both arms at the expected nucleotide positions. FokI cleavage was independent of the spacing between the recognition site and the junction. This new activity of class-IIS ENases may be useful for investigating branched DNA structures.

Cloning, sequencing, and expression of RecA proteins from three distantly related thermophilic eubacteria.

Sequences of the recA genes of the highly divergent thermopholic eubacteria Thermus aquaticus (and Thermus thermophilus), Thermotoga maritima, and Aquifex pyrophilus were determined from fragments derived by polymerase chain reaction (PCR) with degenerate primers and from inverse PCR products obtained using unique primers based on the fragment sequences. The source of the PCR products was verified by Southern hybridization. Complete PCR-derived recA genes were cloned into an expression vector regulated by a temperature-sensitive lambda-repressor, and independently derived clones expressing thermostable recA were selected. DNA sequences were verified to be authentic by direct cycle-sequencing of PCR products and/or sequencing of several clones. In contrast to Escherichia coli RecA protein, all the purified thermophilic RecA proteins exhibited single-stranded DNA-dependent ATPase activity optima above 70 degrees C. Phylogenetic analysis of RecA sequences suggested that the thermophilic RecA proteins were at least as different from one another as were Gram-positive organisms, mesophilic Gram-negative organisms, and cyanobacteria. In spite of substantial sequence divergence, interesting characteristics of the thermostable RecA proteins included increased valine content, common amino acid replacements at two highly conserved sites, and an increase in the calculated isoelectric point of approximately a full pH unit.

Influence of the common human delta-aminolevulinate dehydratase polymorphism on lead body burden.

delta-Aminolevulinate dehydratase (ALAD) is the second enzyme in the heme biosynthesis pathway. ALAD is a zinc metalloenzyme, and its inhibition by lead substitution for zinc is one of the most sensitive indicators of blood-lead accumulation, a measure of recent lead exposure. Stoichiometry calculations indicate that a significant portion of blood lead is stored in ALAD. Human ALAD exhibits a charge polymorphism, with about 20% of Caucasians expressing the rarer ALAD2 allele. Human ALAD1 and ALAD2 cDNAs and the 16-kb ALAD gene have been cloned and sequenced. A simple polymerase chain reaction test has been established and validated for determining ALAD genotypes. Two population studies have indicated that lead-exposed individuals with the ALAD2 allele have blood-lead levels about 10 micrograms/dl greater than similarly exposed individuals carrying only the ALAD1 allele. Ongoing work is directed toward determining the biochemistry underlying the allele-specific accumulation of blood lead, and toward determining the contribution of human ALAD genotype to lead accumulation in other tissues in transgenic mouse models and to final lead deposition in bone in both mouse and man.

Human delta-aminolevulinate dehydratase (ALAD) gene: structure and alternative splicing of the erythroid and housekeeping mRNAs.

Genomic clones containing human delta-aminolevulinate dehydratase (ALAD), the second enzyme in the heme pathway, were isolated, and the entire sequence was determined in both orientations (15,913 bp; GenBank Accession No. X64467). The gene contained two alternative noncoding exons, 1A and 1B, and 11 coding exons, 2-12. Ten Alu-repetitive elements were within the gene, including an inverted repeat that may have resulted from gene conversion. The housekeeping transcript, which included exon 1A and not 1B, was identified in a human adult liver cDNA library, while an erythroid-specific transcript, which contained exon 1B and not 1A, was detected in a human K562 erythroleukemia cDNA library. The promoter region upstream of housekeeping exon 1A was GC-rich and contained three potential Sp1 elements and a CCAAT box. Further upstream, there were three potential GATA-1 binding sites and an AP1 site. The promoter region upstream of erythroid-specific exon 1B had several CACCC boxes and two potential GATA-1 binding sites. To assess the tissue-specific expression of exons 1A and 1B, HeLa and K562 cells were transduced with CAT constructs containing either exon 1A or 1B and their respective upstream promoter region. Two housekeeping CAT constructs, with 450 and 1400 bp upstream of exon 1A, were expressed at similar levels in HeLa cells, whereas the erythroid-specific construct, containing the entire 450-bp promoter region upstream of exon 1B, was not. In contrast, the housekeeping and erythroid constructs were both expressed in K562 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The delta-aminolevulinate dehydratase polymorphism: higher blood lead levels in lead workers and environmentally exposed children with the 1-2 and 2-2 isozymes.

The erythrocyte delta-aminolevulinate dehydratase (ALAD) isozyme phenotypes and the blood lead levels were determined in a population of 202 lead factory workers in Germany and an environmentally exposed population of 1278 children in New York who had elevated free protoporphyrin levels. In both population, individuals with the ALAD 1-2 or 2-2 isozyme phenotype had levels of blood lead statistically higher than those individuals with the ALAD 1-1 isozyme phenotype (lead workers: P greater than 0.004; children: P greater than 0.0001). Homozygotes and heterozygotes for the ALAD2 allele who expressed the 2-2 or 1-2 isozyme phenotype had median blood lead levels that were about 9 to 11 micrograms/dl greater than similarly exposed individuals who were homozygous for the ALAD1 allele. These findings support the hypothesis that the ALAD2 polypeptide binds lead more effectively, and therefore that individuals with the ALAD2 allele may be more susceptible to lead poisoning.

Molecular characterization of the human delta-aminolevulinate dehydratase 2 (ALAD2) allele: implications for molecular screening of individuals for genetic susceptibility to lead poisoning.

The second enzyme in the heme biosynthetic pathway, delta-aminolevulinate dehydratase (ALAD), is a homooctameric protein encoded by a gene localized to human chromosome 9q34. Expression of the two common alleles, ALAD1 (p = .9) and ALAD2 (q = .1), results in a polymorphic enzyme system with three distinct charge isozymes, designated 1-1, 1-2, and 2-2. Individuals heterozygous (2pq = .18) or homozygous (q2 = .01) for the ALAD2 allele have significantly higher blood lead levels than do ALAD1 homozygotes, when exposed to low or high levels of lead in the environment. To investigate the molecular nature of this common polymorphism, total RNA from an ALAD2 homozygote was oligo-dT primed and reverse transcribed, and then the ALAD2 cDNA was amplified, subcloned, and sequenced. Compared with the ALAD1 sequence, the only difference in the ALAD2 cDNA was a G-to-C transversion of nucleotide 177 in the coding region, which created an MspI restriction site. This base substitution predicted the replacement of a positively charged lysine by a neutral asparagine (K59N), an amino acid change consistent with the more electronegative charge of the ALAD-2 subunit. The ALAD1 and ALAD2 alleles were easily detected by amplification of a 916-bp region of genomic DNA and MspI digestion which results in 582- and 511-bp products, respectively. Molecular analysis of 85 ALAD1/ALAD2 heterozygotes and of eight ALAD2 homozygotes revealed no discrepancy between the predicted genotype and the erythrocyte isozyme phenotype, indicating that all the ALAD2 alleles analyzed had the G-to-C transversion.(ABSTRACT TRUNCATED AT 250 WORDS)