Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.)

Genes encoding seven enzymes of the monolignol pathway were independently downregulated in alfalfa (Medicago sativa) using antisense and/or RNA interference. In each case, total flux into lignin was reduced, with the largest effects arising from the downregulation of earlier enzymes in the pathway. The downregulation of l-phenylalanine ammonia-lyase, 4-coumarate 3-hydroxylase, hydroxycinnamoyl CoA quinate/shikimate hydroxycinnamoyl transferase, ferulate 5-hydroxylase or caffeic acid 3-O-methyltransferase resulted in compositional changes in lignin and wall-bound hydroxycinnamic acids consistent with the current models of the monolignol pathway. However, downregulating caffeoyl CoA 3-O-methyltransferase neither reduced syringyl (S) lignin units nor wall-bound ferulate, inconsistent with a role for this enzyme in 3-O-methylation ofS monolignol precursors and hydroxycinnamic acids. Paradoxically, lignin composition differed in plants downregulated in either cinnamate 4-hydroxylase or phenylalanine ammonia-lyase. No changes in the levels of acylated flavonoids were observed in the various transgenic lines. The current model for monolignol and ferulate biosynthesis appears to be an over-simplification, at least in alfalfa, and additional enzymes may be needed for the 3-O-methylation reactions of S lignin and ferulate biosynthesis.     

Characterization of antibodies against methyl-pppN cap structure: plant U3 small nucleolar RNA is recognized by these antibodies.

In eukaryotes, many small nuclear RNAs contain either a trimethylguanosine cap structure of a gamma-monomethyl (me) cap structure. Previously, we reported the characterization of anti-mepppG antibodies which recognize methyl-capped RNAs with G as the initiation nucleotide. We report here the preparation of antibodies against mepppN cap structure. Anti-mepppN antibodies recognized only mepppN from a mixture of mepppN and pppN and immunoprecipitated mepppA-capped U3 small nucleolar RNA from a mixture of cowpea cell RNAs. These anti-mepppN antibodies recognized methylated nucleoside triphosphates (mepppA, mepppC, mepppG and mepppU) with nearly equal efficiency; however, these antibodies did not recognize methyl phosphate or methylated mononucleotides. These antibodies will be useful in the identification and characterization of all methyl-capped RNAs no matter which is the initiation nucleotide.     

Secondary metabolite scale-up to minimize homolog impurity levels

A mutant strain of Streptomyces hygroscopicus was found to produce up to 9.0 units/L of an immunoregulant precursor, immunomycin, with up to 3.5% of a lower homolog impurity under either dual fed-batch or batch conditions. Glycerol and valine were key nutrients influencing productivity and impurity levels. Soybean oil was successfully substituted for glycerol as a carbon source to minimize shot additions to batch culture. The remainder of the production medium was composed largely of defined components with the exception of yeast extract. Valine limitation increased lower homolog formation while decreasing higher homolog formation; excess valine decreased lower homolog formation below 2-3% while increasing higher homolog formation. Higher homolog formation in the presence of valine seemed to be slower than lower homolog formation in the absence of valine. Valine was believed to be the major butyrate precursor; consequently its availability influenced the impurity profile. A preliminary cost analysis suggests that elimination of added valine from the cultivation and replacement of glycerol with soybean oil can result in a 6.6-fold reduction in media costs relative to the original fed-batch process. Copyright 1998 John Wiley & Sons, Inc.     

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.     

Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence

EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5'-CAGCAG-3' transferring a methyl group from S-adenosyl-l-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases.     

Development of a bioconversion process for production of cis-1S,2R-indandiol from indene by recombinant Escherichia coli constructs

Recombinant Escherichia coli cells expressing the toluene dioxygenase (TDO) genes from Pseudomonas putida convert indene to cis-1S,2R-indandiol, a potentially important intermediate for the chemical synthesis of the HIV-1 protease inhibitor, Crixivan. A bioconversion process was developed through optimization of medium composition and reaction conditions at the shake-flask and 23-l fermentor scales. A cis-1,2-indandiol productivity of approx. 1000 mg/l was achieved with construct TDO123, which represents a 50-fold increase over the initial titer. Varying the bioconversion conditions did not change the enantiomeric excess (e.e.) for the 1S,2R enantiomer from about 30%, suggesting that toluene dioxygenase intrinsically converts indene to 1S,2R- and 1R,2S-indandiols at a ratio of 2:1. Further inclusion of the Pseudomonas dehydrogenase gene in construct D160-1 led to the production of chirally pure cis-1S,2R-indandiol (e.e. > 99%) as a result of the selective degradation of the 1R,2S enantiomer, with the overall yield (650 mg/l) proportionally reduced. A single stage process was developed for D160-1 and scaled up to the 23-l fermentor, achieving a cis-1S,2R-indandiol titer of 1200 mg/l. Received: 24 September 1998 / Received revision: 22 January 1999 / Accepted: 31 January 1999     

Malignant human gliomas express an amiloride-sensitive Na+ conductance

Human astrocytoma cells were studied using whole cellpatch-clamp recording. An inward, amiloride-sensitiveNa⁺ current was identified in fourcontinuous cell lines originally derived from human glioblastoma cells(CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures ofcells obtained from glioblastoma multiforme tumors (up to 4 passages).In addition, cells freshly isolated from a resected medulloblastomatumor displayed this same characteristic inward current. In contrast,amiloride-sensitive currents were not observed in normal humanastrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas.The only amiloride-sensitive Na⁺channels thus far molecularly identified in brain are the brain Na⁺ channels (BNaCs). RT-PCRanalyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 inthese tumors and in normal astrocytes. These results indicate that thefunctional expression of amiloride-sensitive Na⁺ currents is a characteristicfeature of malignant brain tumor cells and that this pathway may be apotentially useful target for therapeutic intervention.

     

HIV‐1 gp120 envelope protein modulates proliferation of human glomerular epithelial cells

Glomerular epithelial cells (GEC) have been demonstrated to undergo morphological alterations in human immunodeficiency virus (HIV)‐associated focal glomerulosclerosis. In the present study, we evaluated the effect of HIV‐1 gp120 envelope protein on the growth of cultured human (H) GEC. gp120 protein enhanced (P < 0.001) the proliferation of HGEC at lower concentrations. The mitogenic effect of gp120 protein on HGEC was further confirmed by enhanced accumulation of proliferating nuclear cell antigen (PCNA) by gp120 protein‐treated cells, as compared with control cells. On the contrary, gp120 protein at higher concentrations suppressed (P < 0.001) the growth of HGEC. To evaluate the mechanism of gp120 protein‐induced HGEC growth suppression, we examined the effect of gp120 protein on HGEC apoptosis. gp120 protein at higher concentrations promoted the apoptosis of HGEC. At higher concentrations, gp120 protein also enhanced DNA fragmentation of HGEC. Anti‐gp120 antibody attenuated the proliferative as well as the apoptotic effects of gp120 protein on HGEC. Because protein kinase C as well as tyrosine kinase inhibitors partially inhibited gp120‐induced proliferation, gp120 appears to be activating both the protein kinase C and tyrosine kinase pathways. In addition, gp120 protein at lower concentrations enhanced mRNA expression of c‐fos and at higher concentrations promoted mRNA expression of c‐jun. We conclude that gp120 has a bimodal effect on proliferation of HGEC. This effect may be mediated through the activation of early growth genes. J. Cell. Biochem. 76:61–70, 1999. © 1999 Wiley‐Liss, Inc.