Overexpression of trehalose synthase and accumulation of intracellular trehalose in 293H and 293FTetR:Hyg cells.

A humanized clone containing the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase (otsA/B) has been constructed. Using the Gateway Cloning System (Invitrogen, Inc.), the otsA/B genes have been placed under the control of the CMV promoter (pEXPcmv-otsA/B) or the CMV promoter and the tet operator (pEXP cmv TetO-otsA/B). The pEXPcmv-otsA/B clone has been introduced into 293H cells using LIPOFECTAMINE 2000 and the intracellular concentration of trehalose has been evaluated. The 293H cells accumulate 4-5 microg trehalose/mg dry weight and this concentration increases to 7-10 microg trehalose/mg dry weight if trehalose is included in the growth medium. The pEXPcmv TetO-otsA/B clone has been transfected into 293FTetR:Hyg cells which contain the tet repressor integrated into the genome. When these transfected cells are grown in the absence of tetracycline, no intracellular trehalose is detected. Inclusion of 0.3 microg/ml tetracycline in the growth medium results in the accumulation of 11-14 microg trehalose/mg dry weight, a value which increases to 19-20 microg trehalose/mg dry weight if trehalose is included in the growth medium. The data for the 293FTetR:Hyg cells indicate that intracellular trehalose accumulates in response to the addition of tetracycline. This system will allow us to manipulate the intracellular concentration of trehalose and to evaluate the desiccation tolerance of these cells as a function of intracellular trehalose concentration.

Application of TEV Protease in Protein Production.

In many cases, the analysis of a specific protein is impeded by the inability to purify large amounts of it from a native source. Proteins of interest may be present in minute quantities and/or purification may be plagued with technical problems. Recombinant DNA methodologies have enabled researchers to circumvent some of these limitations by producing and purifying large quantities of protein in a nonnative system. Various systems and strategies have been successfully employed, depending on the specific protein of interest and the desired use of the final end product (antibody production, crystallography studies etc.). This chapter reviews some common methods for the production of recombinant fusion proteins and specifically describes a versatrle method for the removal of affinity tags from recombinant fusions using a highly purified proteinase with an unparalleled degree of specificity. This proteinase, from the genome of tobacco etch virus (TEV), demonstrates specific proteolytic activity under a wide range of parameters (salt, temperature, pH), making it an excellent choice for cleavage of fusion proteins (1,2).

Generation of Recombinant Baculovirus DNA in E.coli Using a Baculovirus Shuttle Vector.

Baculovirus vectors are now widely used to direct the expression of heterologous genes in cultured insect cells and insect larvae. In most cases, heterologous genes placed under transcriptional control of the polyhedrm promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) are abundantly expressed during the late stages of infection. The recombinant proteins are usually soluble and functionally similar to their authentic counterparts (l-7). In the following sections, recent advances in the development of baculovtrus vectors, particularly the baculovn-us shuttle vector system, will be described.

Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.

The methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine (m6A), is catalyzed by a complex multicomponent enzyme. Previous studies suggested that m6A affects the efficiency of mRNA processing or transport, although the mechanism by which this occurs is not known. As a step toward better understanding the mechanism and function of this ubiquitous posttranscriptional modification, we have shown that HeLa mRNA (N6-adenosine)-methyltransferase requires at least two separate protein factors, MT-A and MT-B, and MT-A contains the AdoMet binding site on a 70-kDa subunit (MT-A70). MT-A70 was purified by conventional chromatography and electrophoresis, and was microsequenced. The peptide sequence was used to design a degenerate oligodeoxynucleotide that in turn was used to isolate the cDNA clone coding for MT-A70 from a HeLa cDNA library. Recombinant MT-A70 was expressed as a fusion protein in bacteria and was used to generate anti-MT-A70 antisera in rabbits. These antisera recognize MT-A70 in HeLa nuclear extracts by western blot and are capable of depleting (N6-adenosine)-methyltransferase activity from HeLa nuclear extract, confirming that MT-A70 is a critical subunit of (N6-adenosine)-methyltransferase. Northern blot analysis reveals that MT-A70 mRNA is present in a wide variety of human tissues and may undergo alternative splicing. MT-A70 cDNA probe hybridizes to a 2.0-kilobase (kb) polyadenylated RNA isolated from HeLa cells, whereas it hybridizes to two predominant RNA species (approximately 2.0 kb and 3.0 kb) using mRNA isolated from six different human tissues. Analysis of the cDNA sequence indicates that it codes for a 580-amino acid protein with a predicted MW = 65 kDa. The predicted protein contains sequences similar to consensus methylation motifs I and II identified in prokaryotic DNA (N6-adenosine)-methyltransferases, suggesting the functional conservation of peptide motifs. MT-A70 also contains a long region of homology to the yeast protein SPO8, which is involved in induction of sporulation by an unknown mechanism.

Selective immunoneutralization of the multiple activities of Escherichia coli DNA polymerase I supports the model for separate active sites and indicates a complex 5' to 3' exonuclease.

DNA polymerase I (pol I) from Escherichia coli has three well-defined activities: DNA polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. We have raised monoclonal antibodies to pol I which selectively neutralize each of these three activities, thus supporting the model of separate active sites for each activity, heretofore exclusively demonstrated with proteolytic fragments of pol I. Antibodies from each class could bind pol I in the presence of antibodies of another class, indicating the existence of significant spatial separation between each of the three sites. In addition, several of the neutralizing antibodies were able to distinguish particular activities of the 5'-3' exonuclease. One of them, for example, inhibited the RNase H activity but not the DNase activity. Two other antibodies could, in addition to inhibiting the polymerase and the 3'-5' exonuclease, either stimulate or inhibit the 5'-3' exonuclease depending upon the assay conditions, particularly the ionic strength.

Cyclic AMP-cyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli.

The spf gene of Escherichia coli encodes an unstable 109-nucleotide RNA, spot 42 RNA; the level of this RNA was reduced three- to fivefold when cells were grown in the presence of 3',5'-cyclic AMP (cAMP). We show that this regulation occurs through reduction in transcription and depends on both cAMP and the cAMP receptor protein (CRP) but is independent of the de novo protein synthesis. Through deletion analysis of the spf gene promoter, we have identified sequences that are important in the synthesis of spot 42 RNA. Deletion of sequences upstream of -77 completely eliminated the negative control of cAMP-CRP and resulted in high constitutive levels of transcription. This region contained a sequence that both conformed to the consensus binding site for cAMP-CRP in positively regulated promoters and acted as a cAMP-CRP binding site in a gel retardation assay. Deletion of sequences between positions -77 and -60 greatly reduced the level of transcription in the presence or absence of cAMP-CRP, indicating that at least part of this region is a binding site for a positive-acting transcription factor (or RNA polymerase itself). We propose that the proximity of the two sites defined here allows for the negative control of spf gene transcription by cAMP-CRP. In particular, if only one site at a time can be occupied, the binding of cAMP-CRP would interfere with the binding of a transcription factor.

DNA polymerase I activity in Escherichia coli is influenced by spot 42 RNA.

We have shown that the level of DNA polymerase I (Pol I) activity in Escherichia coli is influenced by the level of a 109-nucleotide RNA, spot 42 RNA. Deletion of the gene for spot 42 RNA results in a 20 to 25% decrease in Pol I activity, as assayed by nucleotide incorporation in cell extracts and a decrease in the ability of cells to grow in the presence of the DNA-alkylating agent methyl methanesulfonate. Also, a physiological reduction of the level of spot 42 RNA, by growth in media containing poor carbon sources, results in a corresponding decrease in Pol I activity. Conversely, overproduction of spot 42 RNA results in a 10 to 15% increase in Pol I activity in vitro. Thus, changes in the amount of spot 42 RNA result in relatively small but significant changes in Pol I activity.

Spot 42 RNA of Escherichia coli is not an mRNA.

Spot 42 RNA of Escherichia coli, a 109-nucleotide RNA that influences the level of DNA polymerase I, has an AUG triplet preceded by a purine-rich potential ribosome-binding site and is followed by a short (14-triplet) potential open reading frame. Although the RNA bound to ribosomes, it did so inefficiently and nonproductively. When fused to lacZ sequences, spot RNA did not support the synthesis of beta-galactosidase. Also, the biological effects of spot 42 RNA were not altered by mutation of the tyrosine UAU codon to the chain termination UAG. We conclude that the effects of spot 42 RNA are mediated by the RNA itself and not by a spot 42 RNA-encoded peptide.

Purine synthesis and catabolism in soybean seedlings : the biogenesis of ureides.

The ureides, allantoin and allantoic acid, are the major nitrogenous substances transported within the xylem of N(2)-fixing soybeans (Glycine max L. Merr. cv Amsoy 71). The ureides accumulated in the cotyledons, roots and shoots of soybean seedlings inoculated with Rhizobium or grown in the presence of 10 millimolar nitrate. The patterns of activity for uricase and allantoinase, enzymes involved in ureide synthesis, were positively correlated with the accumulation of ureides in the roots and cotyledons. Allopurinol and azaserine inhibited ureide production in 3-day-old cotyledons while no inhibition was observed in the roots. Incubation of 4-day-old seedlings with [(14)C]serine indicated that in the cotyledons ureides arose via de novo synthesis of purines. The source of ureides in both 3- and 4-day-old roots was probably the cotyledons. The inhibition of ureide accumulation by allopurinol but not azaserine in 8-day-old cotyledons suggested that ureides in these older cotyledons arose via nucleotide breakdown. Incubation of 8-day-old plants with [(14)C]serine suggested that the roots had acquired the capability to synthesize ureides via de novo synthesis of purines. These data indicate that both de novo purine synthesis and nucleotide breakdown are involved in the production of ureides in young soybean seedlings.