Transposition of plasmid DNA segments specifying hydrocarbon degradation and their expression in various microorganisms

The conjugative TOL plasmid (75 Mdal), specifying biodegradation of xylenes, toluene, and trimethylbenzene derivatives, undergoes dissociation in Pseudomonas aeruginosa PAO to a nonconjugative TOL(*) plasmid (28 Mdal) and a transfer plasmid termed TOLDelta (48 Mdal). The TOL(*) plasmid is rendered transmissible through introduction of a number of conjugative plasmids such as factor K, CAM, and TOLDelta but not by the FP2 derivative pR0271. Transfer of TOL(*) via factor K or TOLDelta is mediated by the formation of plasmid cointegrates; no recombination is observed with CAM. A recombinant RP4-TOL plasmid (76 Mdal), which has lost resistance to tetracycline, has been isolated. The TOL(*) segment can be transposed from this RP4-TOL recombinant plasmid to other antibiotic resistance plasmids such as R702. A segment of DNA, specifying salicylate degradation from SAL plasmid, was transposed onto pAC10, the TOL(*-) derivative of RP4-TOL recombinant plasmid, which has lost resistance to tetracycline but retains the transfer genes of RP4. Transposition of the salicylate degradative genes onto pAC10 results in the loss of kanamycin resistance. It has been possible to isolate SAL(+) segregants from pAC10[unk]SAL transposition derivatives that have lost the pAC10 plasmid. Such segregants harbor the salicylate degradative genes in the form of a nonconjugative plasmid (SAL(*)). Transfer of RP4[unk]TOL(*) or pAC10[unk]SAL(*) transposition derivatives to Escherichia coli, Salmonella typhimurium, Agrobacterium tumefaciens, or Azotobacter vinelandii results in the functional expression of the antibiotic resistance genes but not of the hydrocarbon degradative genes. Such genes, however, are fully expressed on being transferred back to Pseudomonas.     

细胞色素P450 3A4及谷胱甘肽S转移酶A1重组质粒的构建及表达

目的构建重组质粒pBudCE4.1-细胞色素P450 3A4(CYP3A4)-谷胱甘肽S转移酶A1(GSTA1),使CYP3A4和GSTA1可在真核细胞C3A中表达,并且增加其表达量。方法从含有GSTA1和CYP3A4的开放阅读框(ORF)克隆中扩增GSTA1和CYP3A4基因;将片段GSTA1以及载体pBuDCE4.1双酶切后连接并转化,质粒命名为pBuDCE4.1-GSTA1;将片段CYP3A4以及质粒pBuDCE4.1-GSTA1双酶切后连接并转化,所得重组质粒命名为pBudCE4.1-CYP3A4-GSTA1;测序验证重组克隆中目的基因片段的序列信息;构建稳定细胞系,测定CYP3A4活性及GSTA1的表达情况。结果酶切及测序验证双表达重组质粒pBudCE4.1-CYP3A4-GSTA1构建成功,CYP3A4及GSTA1在转染细胞系中的表达量增多。结论构建成的双表达重组质粒pBudCE4.1-CYP3A4-GSTA1符合应用要求。     

Effects of hyaluronic acid on cartilage degradation.

Based on the published literature available so far, it appears that naturally derived hyaluronic acid (HA) and newer formulations available on the market belong to the pharmacologic class of slow-acting drugs for the treatment of osteoarthritis. These compounds seem to have the potential to modulate the painful symptoms of osteoarthritis as well as to improve the function of the osteoarthritis joint. Positive clinical consequences are based on direct and indirect effects of viscosupplementation associated with a normalization of the rheologic properties of the osteoarthritic synovial fluid, decreased inflammation, and end-coating of the pain receptors in the osteoarthritis joint. Few in vivo data exist in humans to support the concept that HA formulations could have a structure-modifying effect on human osteoarthritis cartilage. Animal-based studies have demonstrated positive effects of exogenous HA on pain in the joint, heat shock proteins, and in models of osteoarthritis. Although many promising effects of exogenous HA have been reported, there remains uncertainty as to the effectiveness of reversing cartilage injury and other manifestations of joint diseases with exogenous HA because of difficulties in interpreting and unifying results of these studies. This is due largely to differences of cartilage source in models of joint/cartilage injury, multiple end points, the controls employed, analytical techniques, and the molecular weight of exogenous HA used. There exists a need for uniform agreement as to the choice of injury model, time points of the study, evaluation tools, and source and molecular weight of the HA used if we are to determine whether exogenous application of HA has a truly beneficial role in the reversal of cartilage injury.     

Frequency of δ+27-thalassaemia in Sardinians

Summary To determine the incidence of δ⁺27 thalassaemia in Northern Sardinia we examined blood samples from 750 Sardinian schoolboys by PCR-based molecular analysis. The incidence of δ⁺27 mutation was 1.2% in this study, i.e. twice as high as previously described on the basis of phenotypical studies; the frequency of the β-thalassaemia is 10.5% and their interaction has been calculated at 0.0003. The majority of δ⁺27 carriers are characterized by a HbA₂ level lower than 1.9% and the mean HbA₂ level is significantly lower than in normal subjects. All compound heterozygotes for δ⁺27 and β-thalassaemia show a silent β-thalassaemic phenotype related to normalization of their HbA₂ levels. This study suggests that δ⁺27 thalassaemia should be borne in mind in counselling at-risk couples in which one member has the typical high HbA₂ β-thal trait while the other shows normal or borderline HbA₂ level. In these subjects, PCR-based ECO O 109 I digestion of the δ globin gene allows rapid detection of the δ⁺27 mutation.     

Significance of plasma 7alpha-hydroxy-4-cholesten-3-one and 27-hydroxycholesterol concentrations as markers for hepatic bile acid synthesis in cholesterol-fed rabbits

Plasma 7alpha-hydroxy-4-cholesten-3-one has been used as an index of hepatic bile acid synthesis. The aim of the current study was to ascertain whether the level of this oxysterol reflects hepatic cholesterol 7alpha-hydroxylase activity when plasma cholesterol concentrations are markedly changed. In addition, the relationship of hepatic sterol 27-hydroxylase activity with plasma concentrations of 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid was studied. We used New Zealand white rabbits fed 2% cholesterol for 5 or 10 days and/or constructed bile fistula. Feeding cholesterol markedly increased and bile drainage reduced plasma cholesterol concentrations. Initially, in these models there was no correlation between plasma 7alpha-hydroxy-4-cholesten-3-one concentrations and hepatic cholesterol 7alpha-hydroxylase activities (r = -0.24, n = 10). Cholesterol feeding was associated with downregulated 7alpha-hydroxylase activities, while plasma 7alpha-hydroxy-4-cholesten-3-one concentrations were elevated in the presence of increased plasma cholesterol levels. However, this discrepancy was overcome and significant correlation was observed (r = 0.73, P <.05, n = 10) by expressing 7alpha-hydroxy-4-cholesten-3-one levels relative to cholesterol. In contrast, hepatic sterol 27-hydroxylase activities were not significantly correlated with plasma absolute (r = 0.23, difference not significant [NS], n = 10) nor cholesterol-related levels of 27-hydroxycholesterol (r = -0.13, NS, n = 10), or 3beta-hydroxy-5-cholestenoic acid concentrations (r = 0.30, NS, n = 10). In conclusion, plasma 7alpha-hydroxy-4-cholesten-3-one concentrations reflected hepatic cholesterol 7alpha-hydroxylase activities when the sterol levels were adjusted to plasma cholesterol concentrations in rabbits with hypercholesterolemia. The results suggest that plasma 7alpha-hydroxy-4-cholesten-3-one relative to cholesterol is a better marker for hepatic cholesterol 7alpha-hydroxylase activity than the absolute concentration when hypercholesterolemia is present. In contrast, 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid levels in plasma did not reflect hepatic sterol 27-hydroxylase activities even if the levels were adjusted to plasma cholesterol concentrations.     

Identification of a common nucleotide sequence in the 3''-untranslated region of mRNA molecules specifying inflammatory mediators.

Recently, cDNA sequences have been reported for both human and murine tumor necrosis factor (TNF; cachectin). The coding region of the TNF genes is highly conserved between man and mouse; 80% homology is apparent at the amino acid level. We now observe that a 33-nucleotide sequence, comprised entirely of A and T residues and located in the 3'-untranslated region, is conserved in toto in the murine and human TNF mRNAs. Since the 3'-untranslated region is normally not conserved, we reasoned that this sequence might play a regulatory role. We identified a consensus sequence (TTATTTAT) present in the 3'-untranslated region of both human and mouse TNF mRNAs, as well as the mRNAs encoding human lymphotoxin, human colony stimulating factor, human and mouse interleukin 1, human and rat fibronectin, and most of the sequenced human and mouse interferons. All of these mRNAs, except the lymphotoxin mRNA, lack homology to the TNF mRNAs in the coding region. The consensus sequence is uncommon among mammalian mRNAs in general, but it appears with a frequency greater than chance alone would dictate, suggesting that it may serve a specific regulatory function among the mRNAs in which it is found. It is particularly prevalent among mRNAs encoding proteins related to the inflammatory response.     

Instability of dicentric plasmids in yeast.

Dicentric plasmids containing either two copies of centromere 4 or one copy of centromere 4 and one copy of centromere 3 in the yeast plasmid vector YRp17 were constructed in vitro and introduced into yeast cells by DNA transformation. The resulting colonies were heterogeneous for a mixed population of rearranged plasmids. The rearrangements always involved deletion of one or both centromere sequences originally present on the plasmid. Heterogeneity was due to the continued production of deleted plasmids from a pool of unrearranged dicentric plasmids maintained within some of the yeast cells in the colony. The RAD52 gene product is known to be required for the repair of DNA double-strand breaks in yeast. Transformation of rad52 mutant yeast cells with dicentric plasmids gave rearranged plasmids similar to those observed with RAD+ yeast cells, but the transformation frequency was only 5-10% compared to transformation with monocentric plasmids. Also, the ratio of unrearranged dicentric plasmid to deleted plasmids was greatly reduced in the rad52-transformed cells. These observations are consistent with a model in which centromeric DNA sequences can interact independently with the yeast cell spindle apparatus. Occasional movement of centromeres to opposite poles may result in mechanical breakage of plasmid sequences. Plasmids deleted for one or both centromere sequences can be obtained from these broken molecules and are resistant to further rearrangement.     

Colchicine inhibits epidermal growth factor degradation in 3T3 cells.

Colchicine (2 microM) did not affect the initial rate of association of 125I-labeled epidermal growth factor (125I-EGF) to Swiss 3T3 cells but continued incubation (up to 24 hr) led to an increase in cell-associated radioactivity. The effect is also produced by Colcemid, vinblastine, and podophyllotoxin but not by lumicolchicine. Disruption of microtubules with colchicine does not alter the rate of "down regulation" of EGF receptors, suggesting the binding and internalization of the factor proceed unchanged. However, colchicine markedly decreases the rate of appearance of acid-soluble radioactivity from cells either incubated continuously with 125I-EGF for 24 hr or exposed to the radioactive peptide for only 1 or 3 hr. The results indicate that colchicine decreases the rate of degradation of internalized 125I-EGF. Because antitubulin agents enhance the mitogenic effect of EGF our results suggest that peptide degradation can be dissociated from the long-term biological effect.     

Cul4A targets p27 for degradation and regulates proliferation, cell cycle exit, and differentiation during erythropoiesis

As erythroid progenitors differentiate into precursors and finally mature red blood cells, lineage-specific genes are induced, and proliferation declines until cell cycle exit. Cul4A encodes a core subunit of a ubiquitin ligase that targets proteins for ubiquitin-mediated degradation, and Cul4A-haploinsufficient mice display hematopoietic dysregulation with fewer multipotential and erythroid-committed progenitors. In this study, stress induced by 5-fluorouracil or phenylhydrazine revealed a delay in the recovery of erythroid progenitors, early precursors, and normal hematocrits in Cul4A(+/-) mice. Conversely, overexpression of Cul4A in a growth factor-dependent, proerythroblast cell line increased proliferation and the proportion of cells in S phase. When these proerythroblasts were induced to terminally differentiate, endogenous Cul4A protein expression declined 3.6-fold. Its enforced expression interfered with erythrocyte maturation and cell cycle exit and, instead, promoted proliferation. Furthermore, p27 normally accumulates during erythroid terminal differentiation, but Cul4A-enforced expression destabilized p27 and attenuated its accumulation. Cul4A and p27 proteins coimmunoprecipitate, indicating that a Cul4A ubiquitin ligase targets p27 for degradation. These findings indicate that a Cul4A ubiquitin ligase positively regulates proliferation by targeting p27 for degradation and that Cul4A down-regulation during terminal erythroid differentiation allows p27 to accumulate and signal cell cycle exit.     

Delta 4-3-oxosteroid 5 beta-reductase deficiency: failure of ursodeoxycholic acid treatment and response to chenodeoxycholic acid plus cholic acid.

BACKGROUND--In some infants with liver disease, 3-oxo-delta 4 bile acids are the major bile acids in urine, a phenomenon attributed to reduced activity of the delta 4-3-oxosteroid 5 beta-reductase required for synthesis of chenodeoxycholic acid and cholic acid. These patients form a heterogeneous group. Many have a known cause of hepatic dysfunction and plasma concentrations of chenodeoxycholic acid and cholic acid that are actually greater than those of the 3-oxo-delta 4 bile acids. It is unlikely that these patients have a primary genetic deficiency of the 5 beta-reductase enzyme. AIMS--To document the bile acid profile, clinical phenotype, and response to treatment of an infant with cholestasis, increased plasma concentrations of 3-oxo-delta 4 bile acids, low plasma concentrations of chenodeoxycholic acid and cholic acid, and no other identifiable cause of liver disease. PATIENTS--This infant was compared with normal infants and infants with cholestasis of known cause. METHODS--Analysis of bile acids by liquid secondary ionisation mass spectrometry and gas chromatography-mass spectrometry. RESULTS--The plasma bile acid profile of the patient was unique. She had chronic cholestatic liver disease associated with malabsorption of vitamins D and E and a normal gamma-glutamyltranspeptidase when the transaminases were increased. The liver disease failed to improve with ursodeoxycholic acid but responded to a combination of chenodeoxycholic acid and cholic acid. CONCLUSION--Treatment of primary 5 beta-reductase deficiency requires the use of bile acids that inhibit cholesterol 7 alpha-hydroxylase.     

Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation.

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by (1)H NMR as a 4-methyl-24, 25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.     

Chemoattractants stimulate degradation of methylated phospholipids and release of arachidonic acid in rabbit leukocytes.

When rabbit peritoneal leukocytes were treated with chemoattractants such as fMet-Leu-Phe, an apparent decrease of [3H]methyl incorporation into the lipid fraction from L-[methyl-3H]methionine was observed. This decrease was a result of increased degradation of methylated phospholipids, not of decreased synthesis. Chemotactic peptides did not affect the metabolism of the phospholipids in which [methyl-14C]choline was incorporated. The disappearance of the [3H]methyl group was associated with the release of [1-14C]arachidonic acid from phospholipids prelabeled with these compounds. These findings suggested the activation by chemoattractants of phospholipase A2, an enzyme that removes an unsaturated fatty acid from phospholipids. The order of potency of chemoattractants for the stimulated degradation of phospholipids was in good agreement with that for chemotaxis. Mepacrine (quinacrine) and hydrocortisone inhibited and a phorbol ester enhanced both chemotaxis and phospholipase A2 activity. These results, taken together, suggest close association of the metabolism of methylated phospholipids with chemotaxis in rabbit peritoneal leukocytes.     

Construction and properties of chimeric plasmids in Bacillus subtilis.

Antibiotic resistance chimeric plasmids have been constructed by in vitro enzymatic manipulation and introduced into Bacillus subtilis by transformation. The parental plasmids used had been introduced into B. subtilis from Staphylococcus aureus by transformation. Of the seven recombinant plasmids constructed using restriction endonucleases, one was made using EcoRI, another using Hpa II, and five with Xba I (from Xanthomonas badrii), demonstrating the utility of the latter enzyme for molecular cloning experiments. Although all of the recombinant plasmids we have made replicate and express their antibiotic resistance characters, three of them have suffered a loss of DNA, either in vitro or, more likely, in vivo. The deletion event in all cases involved one of the two termini used to join the parental plasmids. The plasmid chimeras reported in this paper should prove useful for the study of plasmid replication, incompatibility, and recombination. In addition, the utility of the B. subtilis system for molecular cloning has been clearly illustrated.     

Mitotic and meiotic stability of linear plasmids in yeast.

Circular recombinant DNA plasmids that contain autonomously replicating sequences (ARSs) are maintained in extrachromosomal form in transformed yeast cells. However, these plasmids are unstable, being rapidly lost from cells growing without selection. Although the stability of such a plasmid can be increased by the presence of yeast centromere DNA (CEN), even CEN plasmids are lost at a high rate compared to a bona fide yeast chromosome. Natural yeast chromosomes are linear molecules; therefore, we have asked if linearization can improve the stability of recombinant DNA plasmids. Linear plasmids with and without yeast CENs were constructed in vitro by using termini from the extrachromosomal ribosomal DNA (rDNA) of the ciliated protozoan Tetrahymena thermophila as "telomeres." These linear plasmids transformed yeast at high frequency and were maintained as linear extrachromosomal molecules during mitotic growth. Moreover, linear plasmids containing CENs were also transmitted through meiosis: these plasmids segregate predominantly 2+:2- at the first meiotic division, indicating that Tetrahymena rDNA termini can provide telomere function during yeast meiosis. Linear plasmids without CENs were about as stable in mitosis as the comparable circular plasmid. Thus, the Tetrahymena rDNA termini have no marked positive or negative effect on the mitotic stability of ARS plasmids. However, linear plasmids containing CENs are three to four times less stable in mitotic cells than circular CEN plasmids. This decrease in stability is not due to a functional change in the centromere itself; rather, linearization of a CEN plasmid has a direct detrimental effect on its mitotic stability. These results may reflect the existence of spatial constraints on the positions of centromeres and telomeres, constraints which must be satisfied to achieve stable segregation of chromosomes during mitosis.     

RecA-independent recombination at gamma delta termini and at IS3 producing inverted repetition in F'' plasmids.

Two F' plasmids isolated independently from a recA- strain of Escherichia coli and containing identical deletion end points and identical associated inverted duplications are described. In these plasmids, DNA of F plasmid from the IS3 element alpha 1 beta 1 up to the transposon gamma delta is duplicated in inverted orientation, and a 63-kilobase-pair segment from the chromosomal DNA of the plasmid is deleted. One deletion terminus is the chromosomal IS3 alpha 4 beta 3 carried by the parental plasmid, ORF203. It is proposed that these structures resulted from interduplex strand exchanges that occurred at the ends of the movable element gamma delta and at IS3. This indicates that the 35-base-pair gamma delta termini can participate in genome rearrangements by mechanisms that are distinct from complete transposition mechanisms.     

Characterization of genomic clones specifying rabbit alpha- and beta-ventricular myosin heavy chains.

We have isolated gene sequences coding for the alpha- and beta-myosin heavy chains (HC) of rabbit ventricular muscle. A rabbit genomic library was screened with previously characterized cDNA clones specifying part of the light meromyosin and the entire subfragment 2 portion of alpha- and beta-myosin HCs, as well as with a clone containing the 3' nontranslated sequences of the alpha-myosin HC mRNA. Seven strongly hybridizing clones were analyzed in detail. One genomic clone encoded all of the 3' nontranslated sequences of an alpha-cDNA clone and, therefore, contained the 3' end of the alpha-myosin HC gene. Electron microscopic heteroduplex analysis and DNA sequence analysis showed that this clone overlapped a second genomic clone providing more than 25 kilobase pairs of the alpha-myosin HC gene. The exons within this region corresponded to approximately equal to 85% of the mRNA and were separated by at least 28 introns. A clone for the beta-myosin HC gene was also identified by Southern blot hybridization, by heteroduplex mapping, and by comparing the DNA sequence of a subfragment 2 exon to sequences of the alpha- and beta-cDNA clones. The introns of the alpha- and beta-myosin HC genes were in the same position but showed marked variation in length. These results conclusively showed that the alpha- and beta-myosin HCs are products of separate genes.