PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods.

This study compared different methods of direct DNA extraction and purification from a silt loam soil and investigated the relationship between DNA quantity and sequence diversity. Five extraction methods and four purification techniques were investigated. Quantities of DNA extracted were between 3.4+/-0.55 and 54.3+/-8.18 &mgr;g g(-1) (dry wt) of soil with OD(260)/OD(230) purity ratios between 0.80 and 1.15. Analysis of sequence diversity in all extracts was conducted using PCR-single strand conformation polymorphism (SSCP). Profiles generated using universal 16S rDNA primers (Com1/Com2) were found to be identical when used to amplify 16S rDNA extracted directly from soil. The genus Pseudomonas was targeted in order to reduce profile complexity, which was apparent when using universal 16S rDNA primers, and which hindered direct comparison of sequence diversity. A Pseudomonas culture library and non-cultured Pseudomonas 16S rDNA genes were used to provide a background count of Pseudomonas operational taxonomic units present in the soil. Cloning and sequencing of amplicons generated using a Pseudomonas-specific (Ps-for) and a universal 16S rDNA (Com2) primer, coupled with nested amplification (Com1/Com2 amplification from Ps-for/Ps-rev amplicons), used in conjunction with SSCP, revealed that environmental contaminants co-extracted with DNA, such as humic acid, significantly reduced primer specificity. SSCP was sensitive enough to reveal template bias in different primer sets. PCR-restriction fragment length-SSCP of Pseudomonas 16S rDNA amplified from soil-extracted DNA revealed distinct differences in sequence representation between extraction methods and showed that greater DNA yield is not synonymous with higher sequence diversity. We, therefore, suggest that DNA extractions from soil should be evaluated not only in terms of quantity and purity, but also in terms of the sequence diversity present. SSCP proved to be a valuable tool for the assessment of the methodologies commonly used in PCR-mediated microbial ecology studies.

Biodegradation of diethyl phthalate in soil by a novel pathway.

Biodegradation of diethyl phthalate (DEP) has been shown to occur as a series of sequential steps common to the degradation of all phthalates. Primary degradation of DEP to phthalic acid (PA) has been reported to involve the hydrolysis of each of the two diethyl chains of the phthalate to produce the monoester monoethyl phthalate (MEP) and then PA. However, in soil co-contaminated with DEP and MeOH, biodegradation of the phthalate to PA resulted in the formation of three compounds, in addition to MEP. These were characterised by gas chromatography-electron ionisation mass spectrometry and nuclear magnetic resonance as ethyl methyl phthalate, dimethyl phthalate and monomethyl phthalate, and indicated the existence of an alternative pathway for the degradation of DEP in soil co-contaminated with MeOH. Transesterification or demethylation were proposed as the mechanisms for the formation of the three compounds, although the 7:1 ratio of H(2)O to MeOH means that transesterification is unlikely.

Bioavailability of 2,4-D sorbed to a chlorite-like complex.

An Al(OH)x-montmorillonite (chlorite) complex (AM18) was prepared and 2,4-dichlorophenoxyacetic acid (2,4-D) sorbed to saturation. After several washing cycles the 'strongly sorbed' 2,4-D was 507 micrograms g-1 AM18. The bioavailability of sorbed 2,4-D was assessed in a minimal salts medium with the AM18-2,4-D as the sole C and energy source. Over a 28-day period a Pseudomonas sp. degraded 23% more of the sorbed 2,4-D than could be accounted for by desorption from AM18 in the non-inoculated controls. Possible explanations for the increase in bioavailability are presented.

Fate of phenanthrene, pyrene and benzo[a]pyrene during biodegradation of crude oil added to two soils.

The release of 14CO2 from 9-[14C]phenanthrene, 4,5,9,10-[14C]pyrene and 7-[14C]benzo[a]pyrene, added to Brent/Fortes crude oil and mixed into a pristine sand soil (0.40% organic C) and a pristine organic soil (22.9% organic C), was determined. After 244 days at 25 degrees C, 11.1 +/- 3.5% (sand) and 17.1 +/- 0.30% (organic) phenanthrene-14C and 9.77 +/- 2.8% (sand) and 5.86 +/- 1.4% (organic) benzo[a]pyrene-14C was released. After 210 days, 3.65 +/- 0.5% (sand) and 4.43 +/- 0.33% (organic) pyrene-14C was released. Inoculation of these two soils with DC1 and PD2 (bacteria capable of accelerating the phenanthrene and pyrene mineralisation in soil in the absence of crude oil) either at day 0 or after release as 14CO2 by indigenous degraders had ceased, failed to increase or initiate further mineralisation. Thus, aged PAH residues were non-bioavailable to these metabolically competent degrading microorganisms. At the end of the first period of incubation (210 days or 244 days), the total aromatic hydrocarbons recovered using Soxhlet extraction was 0.18% (sand) and 42.8% (organic) compared with approximately 100% from bio-inhibited soils. This confirmed that the indigenous microbiological activity not only caused a limited amount of PAH mineralisation but also reduced the extractability of residues, possibly due to the generation of metabolites which were chemisorbed and bound (and non extractable) in 'aged' soils.

Bioavailability and biodegradation of polycyclic aromatic hydrocarbons in soils.

Inoculation of soil with bacteria (a Gram-negative rod [PD2] and a 4-membered consortium [DC1]) accelerated mineralisation of phenanthrene and pyrene (but not naphthalene) added individually to a pristine sand and a pristine organic soil. The half-life of naphthalene was 3.5 days in both soils whether inoculated or non-inoculated. However, the half-life of phenanthrene decreased from 86 days in non-inoculated sand soil and 80 days in the non-inoculated organic soil to 3.6 days in the sand and 3.1 days in organic soil when inoculated with PD2, and to 6.6 days in the sand and 8.7 days in the organic soil when inoculated with DC1. Phenanthrene mineralisation ceased after 23 days in DC1-inoculated soil and was 71.3 +/- 3.6% (sand) and 63.3 +/- 2.8% (organic). This compared with 96.8 +/- 3.8% (sand) 102.8 +/- 2.5% (organic) after 8 days in PD2-inoculated soil. Inoculation with DC1 (but not PD2) also accelerated mineralisation of pyrene, where the half-life decreased from 155 days to 18 days in the sand soil, and from 216 days to 33 days in organic soil.

An essential cell division gene of Drosophila, absent from Saccharomyces, encodes an unusual protein with tubulin-like and myosin-like peptide motifs.

Null mutations at the misato locus of Drosophila melanogaster are associated with irregular chromosomal segregation at cell division. The consequences for morphogenesis are that mutant larvae are almost devoid of imaginal disk tissue, have a reduction in brain size, and die before the late third-instar larval stage. To analyze these findings, we isolated cDNAs in and around the misato locus, mapped the breakpoints of chromosomal deficiencies, determined which transcript corresponded to the misato gene, rescued the cell division defects in transgenic organisms, and sequenced the genomic DNA. Database searches revealed that misato codes for a novel protein, the N-terminal half of which contains a mixture of peptide motifs found in alpha-, beta-, and gamma-tubulins, as well as a motif related to part of the myosin heavy chain proteins. The sequence characteristics of misato indicate either that it arose from an ancestral tubulin-like gene, different parts of which underwent convergent evolution to resemble motifs in the conventional tubulins, or that it arose by the capture of motifs from different tubulin genes. The Saccharomyces cerevisiae genome lacks a true homolog of the misato gene, and this finding highlights the emerging problem of assigning functional attributes to orphan genes that occur only in some evolutionary lineages.

Genetic counseling of isolated carriers of Duchenne muscular dystrophy.

It has recently become possible to detect female carriers of Duchenne muscular dystrophy with no affected male relative in the family. These "isolated carriers" represent about 10% of women with high serum creatine phosphokinase (CPK) levels and clinical evidence of a muscle disease. Most isolated carriers ascertained by clinical and/or CPK levels and diagnosed by dystrophin immunostaining of muscle biopsy show symptoms of a muscular dystrophy, and often carry the diagnosis of recessive "limb-girdle muscular dystrophy" prior to dystrophin analysis. It has been difficult to offer genetic counseling and prenatal diagnosis for Duchenne muscular dystrophy in the families of these isolated carriers, largely due to the difficulty in determining which of the dystrophin alleles segregating in the family harbors the mutation in the heterozygote. Here we report genetic counseling of three isolated carriers and their families. In two cases, prenatal diagnosis of at-risk pregnancies was conducted. We determined X inactivation patterns and inheritance of X chromosomes in each family, and used this information to define the at-risk dystrophin gene. In all three families, the mutation was a de novo event, two in the paternal germ-line, and one in the maternal germ-line. In each case we show that sibs of the heterozygous woman are at population risk, while pregnancies of each propositus are at high risk. Our results show that accurate genetic counseling and prenatal diagnosis can be offered to these families.

Reflectance spectroscopy of ferric sulfate-bearing montmorillonites as Mars soil analog materials.

Spectroscopic analyses have shown that smectites enhanced in the laboratory with additional ferric species exhibit important similarities to those of the soils on Mars. Ferrihydrite in these chemically treated smectites has features in the visible to near-infrared region that resemble the energies and band strengths of features in reflectance spectra observed for several bright regions on Mars. New samples have been prepared with sulfate as well, because S was found by Viking to be a major component in the surface material on Mars. A suite of ferrihydrite-bearing and ferric sulfate-bearing montmorillonites, prepared with variable Fe3+ and S concentrations and variable pH conditions, has been analyzed using reflectance spectroscopy in the visible and infrared regions, Mössbauer spectroscopy at room temperature and 4 K, differential thermal analysis, and X-ray diffraction. These analyses support the formation of ferrihydrite of variable crystallinity in the ferrihydrite-bearing montmorillonites and a combination of schwertmannite and ferrihydrite in the ferric sulfate-bearing montmorillonites. Small quantities of poorly crystalline or nanophase forms of other ferric materials may also be present in these samples. The chemical formation conditions of the ferrihydrite-bearing and ferric sulfate-bearing montmorillonites influence the character of the low temperature Mössbauer sextets and the visible reflectance spectra. An absorption minimum is observed at 0.88-0.89 micrometers in spectra of the ferric sulfate-bearing samples, and at 0.89-0.92 micrometers in spectra of the ferrihydrate-bearing montmorillonites. Mössbauer spectra of the ferric sulfate-bearing montmorillonites indicate variable concentrations of ferrihydrite and schwertmannite in the interlaminar spaces and along grain surfaces. Dehydration under reduced atmospheric pressure conditions induces a greater effect on the adsorbed and interlayer water in ferrihydrite-bearing montmorillonite than on the water in ferric sulfate-bearing montmorillonite. Reflectance spectra of ferric sulfate-bearing montmorillonite include a strong 3-micrometers band that is more resistant to dry atmospheric conditions than the 3-micrometers band in spectra of similarly prepared ferrihydrite-bearing montmorillonites.

Site-directed mutagenesis of putative GTP-binding sites of yeast beta-tubulin: evidence that alpha-, beta-, and gamma-tubulins are atypical GTPases.

The exchangeable GTP-binding site on beta-tubulin has been extensively studied, but the primary sequence elements which form the binding site on beta-tubulin remain unknown. We have used site-directed mutagenesis of the single beta-tubulin gene of Saccharomyces cerevisiae to test a model for the GTP-binding site on beta-tubulin, which was based on sequence comparisons with members of the GTPase superfamily [Sternlicht, H., Yaffe, M.B., & Farr, G. W. (1987) FEBS Lett. 214, 226-235]. We analyzed the effects of D295N, N298K, and N298Q mutations in a proposed base-binding motif, 295DAKN298, on tubulin-GTP binding and on nucleotide-binding specificity. We also examined the effects of a D203S mutation in a putative phosphate-binding region, 203DNEA206, on nucleotide binding affinity, on the assembly-dependent tubulin GTPase activity in vitro, and on the dynamic properties of individual "mutant" microtubules in vitro. The effects of the mutations on cell phenotype and on microtubule polymerization in cells were also measured. The results do not support the proposal that the 203DNEA206 and 295DAKN298 [corrected] motifs are cognate to motifs found in GTPase superfamily members. Instead, the data argue that the primary sequence elements of beta-tubulins that interact with bound nucleotide, and presumably also those of the alpha- and gamma-tubulin family members, are different from those of "typical" GTPase superfamily members, such as p21ras. The GTPase superfamily should thus be broadened to include not just the typical GTPases that show strong conservation of primary sequence consensus motifs (GxxxxGK, T, DxxG, NKxD) [corrected] but also "atypical" GTPases, exemplified by the tubulins and other recently identified GTPases, that do not show the consensus motifs of typical GTPases and which also show no obvious primary sequence relationships between themselves. The tubulins and other atypical GTPases thus appear to represent convergent solutions to the GTP-binding and hydrolysis problem.

In vitro assembly of microtubule protein with GTP and 2'dGTP: kinetic evidence for a preassembly conformational change.

The assembly of chick brain microtubule protein in a NaCl-supplemented buffer has been examined with respect to nucleation and the subsequent elongation as a function of the nucleotide (GTP vs 2'dGTP), and the protein and nucleotide concentrations. The kinetics suggest that unassembled tubulin can exist in two conformational states (termed Tu1,GTP and Tu2,GIP when GTP is bound to the exchangeable site), with Tu1,GTP contributing to nucleation and Tu2,GTP participating in elongation. The extent of self-nucleation is proposed to be determined, in part, by the rate constant governing this conformational change. This analysis contrasts with that of earlier studies, which concluded that the number of subunits interacting to form an effective nucleus could be estimated from the dependency of self-nucleation on the initial concentration of unassembled tubulin.

The phosphatidylinositol-binding site of microtubule-associated protein MAP2.

Recent evidence [Surridge and Burns, Biochemistry (1994) 33, 8051-8057] on the interaction of native and recombinant tau, recombinant MAP2c, and native MAP2 with vesicles prepared from phosphatidylinositol (PtdIns) and other phospholipids demonstrate that MAP2 differs from MAP2c and from tau in having a high-affinity PtdIns-binding site. The location of this site within the MAP2-specific insert peptide, coupled with considerations of the nature of the MAP2 tubulin-binding site, suggests that PtdIns-binding induces a conformational change which alters the MAP2 tubulin-binding domain. Furthermore, the restricted cellular distribution of MAP2 implies that the MAP2:PtdIns interaction may play a central role in modulating the dendritic cytoskeleton.

Identification of two new members of the tubulin family.

Analysis of the delta- and epsilon-tubulin sequences indicates that they both consist of two structural domains of which the N-terminal domain can bind to alpha/beta heterodimers while the C-terminal domain probably binds to a non-tubulin protein. Both additional tubulins probably bind GTP but lack GTPase activity, while their synthesis requires the TCP1 chaperonine but is not autoregulated. Although these properties resemble those of gamma-tubulin, the low sequence identity (Table I) demonstrates that the gamma-, delta-, and epsilon-proteins should be classed as different members of the tubulin family. The identification of these additional members is unexpected. Examination of the cellular expression and distribution of the delta- and epsilon-tubulins, and whether other organisms contain homologous genes, may reveal further features of the eukaryotic cytoskeleton.

The difference in the binding of phosphatidylinositol distinguishes MAP2 from MAP2C and Tau.

The interactions of bovine brain MAP2 and tau, recombinant murine MAP2C, and recombinant human tau with phosphatidylinositol vesicles yield apparent Kd values of 51 +/- 6 nM, 2.4 +/- 0.6 microM, 1.4 +/- 0.1 microM, and 1.6 +/- 0.2 microM, respectively. Examinations of the binding of MAP2 and/or MAP2C to phosphatidylcholine vesicles doped with phosphatidylinositol or to phosphatidylserine vesicles and of thrombin-digested MAP2C to phosphatidylinositol vesicles demonstrates that the observed high affinity of the MAP2: phosphatidylinositol binding is due to the contributions of two separate interactions. A low-affinity site (Kd = 1.5-2.5 microM) is located within the C-terminal domain and affects the nonspecific interaction of MAP2, MAP2C, and tau with anionic phospholipids. The second site, with an apparent Kd of 221 +/- 25 nM, is located within the MAP2-specific peptide, which is eliminated from MAP2C by differential gene splicing. It is proposed that the high affinity of MAP2 for phosphatidylinositol contributes to the spatial and temporal regulation of the dendritic cytoskeleton.

Functional role of a consensus peptide which is common to alpha-, beta-, and gamma-tubulin, to actin and centractin, to phytochrome A, and to the TCP1 alpha chaperonin protein.

The TRiC (TCP1 Ring Complex) chaperonin complex participates in the functional folding of actin, centractin, alpha-, beta-, gamma-tubulin, and phytochrome. Each of the cytoskeletal proteins contain a peptide, RK(A,C,T)F/KRAF, located towards the C-terminus, which is homologous to a TCP1 alpha peptide, while the equivalent phytochrome peptide (RLKAF in certain isoforms) is very similar to the KLRAF peptide of TCP1 alpha. We propose that this TCP1 alpha peptide binds to the nascent polypeptides as they emerge from the ribosome, that this binding restricts the folding pathway, and that the TCP1 alpha peptide is subsequently displaced by the synthesis of the consensus peptide. This hypothesis is strongly supported by the crystallographic structure of actin.

The highly divergent alpha- and beta-tubulins from Dictyostelium discoideum are encoded by single genes.

As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dictyostelium alpha- and beta-tubulins, as well as the Dictyostelium alpha-tubulin gene. Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete alpha-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described. Single alpha- and beta-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the alpha- and beta-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium alpha- and beta-tubulins. These antisera recognize one alpha- and two beta-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.