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 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. The transcriptional control of a number of genes is mediated by the binding of cAMP to the cAMP receptor protein (CRP) (for reviews, see references 6 and 25). Often the binding of the cAMP-CRP complex to specific sequences in the DNA results in the stimulation of transcription; however, the expression of several genes has been shown to be negatively regulated, apparently by the inhibition of transcription initiation (1,2,12,17,23,27,28).In (Difco), and 5 g of NaCl (14). When present, ampicillin and chloramphenicol were at 200 ,ug/ml, kanamycin was at 12.5 ,ug/ml, streptomycin was at 115 ,ug/ ml, and rifampin was at 50 ,ug/ml, unless otherwise stated in the text.Plasmid and strain construction. A series of deletions in the spf gene promoter were constructed by using the unidirectional exonuclease protocol of . A 289-base-pair Sau3A fragment of pRD6A (18) containing the spf gene (from positions -161 to + 128) was subcloned into the BamHI site of pGEM-1. The resulting plasmid was digested with PstI and XbaI and incubated with exonuclease III for 1 to 10 min (25°C) and then with exonuclease VII and the Klenow fragment of DNA polymerase I before religation. The end points of the deletions were determined by sequencing (22) (see Fig. 3). The spf promoter deletion genes were then digested with Hindlll and treated with the Klenow fragment of DNA polymerase I, and BglII linkers were added. The BglII-EcoRI fragment containing the spf promoter deletion genes was subcloned into the BamHI-EcoRI sites of pJD2001R, a derivative of pJD2001 (16) in which the unique SalI site 3' of the Kmr gene was converted to an EcoRI site by addition of an EcoRI...
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.
We have shown that the level of DNA polymerase I (Pol I) activity in Eschenichia 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.DNA polymerase I (Pol I) of Escherichia coli functions both in the synthesis of DNA and in the repair of damage caused by a variety of chemical agents such as methyl methanesulfonate (MMS) and physical agents such as UV light (5, 13). Pol I synthesis does not appear to be autogenously regulated, nor is the rate of transcription ofpolA, the gene encoding Pol l, affected by DNA damage (28).Located just 150 nucleotides downstream of the translation stop codon of the polA gene in the E. coli chromosome is a gene called spf, which encodes a 109-nucleotide RNA, spot 42 RNA (9). Spot 42 RNA is moderately abundant (100 to 200 molecules per cell) and has a half-life of 12 to 13 min (21, 22); accumulation of this RNA in vivo is negatively regulated by cyclic AMP and cyclic AMP receptor protein (D. A. Polayes, P. W. Rice, and J. E. Dahlberg, submitted for publication). To date, the biological role of spot 42 RNA has been unclear. Deletion of the spf gene is not lethal, but results in a slight growth impairment of one strain under particular conditions (7). In contrast, overproduction of spot 42 RNA has adverse effects on the growth of most strains of E. coli (19,20).Because of the proximity of the spf and polA genes, we questioned whether they or their products might influence each other. In particular, we studied the influence of spot 42 RNA on the level of Pol I activity in cells, as assayed in extracts or by the ability of cells to survive treatment with MMS. In the experiments presented in this report, we showed that deletion of the spf gene results in a decrease in Pol I activity, as assayed both in vivo and in vitro, and that overproduction of spot 42 RNA results in an increase in Pol I activity. (18). When present, ampicillin was used at 200 ,ug/ml, tetracycline was used at 15 ,ug/ml, and kanamycin was used at 12.5 ,ug/ml, unless otherwise stated in the text. Strains and plasmids. The E. coli K-12 strains used (1) are described in Table 1, and the plasmids used for linear transformations into JC7623 are outlined in Fig. 1. The point mutation in JED2010 was constructed by site-directed mutagenesis (29). To promote integration by homologous recombination, the DNA to be inserted (the spf gene and the 1.2-kilobase-pair [kbp] kanamycin resista...
ABSTRACrThe ureides, allantoin and allantoic acid, are the major nitoge us substances transported within the xylem of Nrfixing soybeans (Glycine max L. Meff. cv Amsoy 71). The ureides accumulated in the cotyledons, roots and shoots of soybean seedlings inoculated with Rhizobiwm 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 4day-old seedlings with 4qClserine indicated that in the cotyledons ureides arose via de novo synthesis of purines. The source of ureides in both 3-and 4day-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 1'4Ciserine 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. tration may result from (a) de novo synthesis of purines with subsequent metabolism to ureides, or (b) the degradation of preexisting nucleotides to form ureides. In either case the activity ofboth uricase and allantoinase, two enzymes involved in ureide synthesis via purine degradation, should be observed. The site of ureide synthesis can be determined by examining the patterns of activity of uricase and allantoinase.The pathway or ureide synthesis can be investigated by using the inhibitors azaserine and allopurinol. Azaserine blocks the transfer of the amide-N of glutamine to formylglycinamide ribonucleotide (30). Allopurinol inhibits the enzyme xanthine dehydrogenase (8). Thus, azaserine is an inhibitor of de novo purine synthesis while allopurinol blocks purine degradation. Fujihara and Yamaguchi (15) found no difference in the amount of ureides in the roots of 3-d-old soybeans after treatment with azaserine. They concluded that de novo synthesis of purines is not involved in ureide production in young seedlings. However, no data were presented for the effects of azaserine on the levels of ureides in the cotyledons.In this paper we examine the pattern of ureide accumulation in the developing soybean seedling. The role of de novo synthesis ofpurines in the production of ureides is addressed by examining the effect of inhibitors of purine metabolism on ureide synthesis and by measuring the incorporation of label from ['4C]serine into purines, allantoin and allantoic acid.The ureides, allantoin and allantoic acid, are the major xylary nitrogenous substances of soybeans grown in association with the bacterium, Rhizobium japonicum (17,20,25). The presence of ureides in the xylem stream has been linked to ...
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).
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