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R P Perry

Publications and source records attributed to R P Perry.

At least 19 recordsLinked to original sources
PubMed1991

Delta, a transcription factor that binds to downstream elements in several polymerase II promoters, is a functionally versatile zinc finger protein.

The promoters of several eukaryotic genes transcribed by RNA polymerase II contain elements located downstream of the transcriptional start site. To gain insight into how these elements function in the formation of an active transcription complex, we have cloned and sequenced the cDNA that encodes delta, a protein that binds to critical downstream promoter elements in the mouse ribosomal protein rpL30 and rpL32 genes. Our results revealed that the delta protein contains four C-terminal zinc fingers, which are essential for its DNA binding capability and a very unusual N-terminal domain that includes stretches of 11 consecutive negatively charged amino acids and 12 consecutive histidines. The sequence of the delta protein was found to be essentially identical to a concurrently cloned human transcription factor that acts both positively and negatively in the context of immunoglobulin enhancers and a viral promoter. Our structural modeling of this protein indicates properties that could endow it with exquisite functional versatility.

Amino Acid Sequence↗
PubMed1991

Oligopyrimidine tract at the 5' end of mammalian ribosomal protein mRNAs is required for their translational control.

Mammalian ribosomal protein (rp) mRNAs are subject to translational control, as illustrated by their selective release from polyribosomes in growth-arrested cells and their underrepresentation in polysomes in normally growing cells. In the present experiments, we have examined whether the translational control of rp mRNAs is attributable to the distinctive features of their 5' untranslated region, in particular to the oligopyrimidine tract adjacent to the cap structure. Murine lymphosarcoma cells were transfected with chimeric genes consisting of selected regions of rp mRNA fused to non-rp mRNA segments, and the translational efficiency of the resulting chimeric mRNAs was assessed in cells that either were growing normally or were growth-arrested by glucocorticoid treatment. We observed that translational control of rpL32 mRNA was abolished when its 5' untranslated region was replaced by that of beta-actin. At the same time, human growth hormone (hGH) mRNA acquired the typical behavior of rp mRNAs when it was preceded by the first 61 nucleotides of rpL30 mRNA or the first 29 nucleotides of rpS16 mRNA. Moreover, the translational control of rpS16-hGH mRNA was abolished by the substitution of purines into the pyrimidine tract or by shortening it from eight to six residues with a concomitant cytidine----uridine change at the 5' terminus. These results indicate that the 5'-terminal pyrimidine tract plays a critical role in the translational control mechanism. Possible factors that might interact with this translational cis regulatory element are discussed.

Animals↗
PubMed1991

Cell-free transcription of a mouse ribosomal-protein-encoding gene: the effects of promoter mutations.

The mouse ribosomal protein-encoding gene, rpS16, was accurately transcribed in vitro with a high-efficiency nuclear extract prepared from HeLa cells. An analysis of the relative activities of rpS16 templates containing deletions or deleterious mutations of various promoter elements indicated that the in vitro transcription system can recognize all of the promoter elements that were previously identified by in vivo transfection experiments. The importance of a polypyrimidine initiator, which spans the cap site, and an element termed C, which is located in the region conventionally occupied by a TATA-box, was also assessed with an appropriate set of mutant templates. In agreement with earlier in vivo studies, our in vitro results indicated that the initiator is the primary determinant for selecting the transcription start point. Interestingly, however, the in vitro system exhibited a strong bias for features that are not present in the natural rpS16 gene. Mutant templates that contained a purine rather than a pyrimidine at or near the cap site or that had the C element replaced by a canonical TATA-box were six- to tenfold more active than the wild-type (wt) rpS16 gene in vitro system, whereas in vivo, these mutants were expressed equivalently to the wt gene. A double mutant containing both a cap site substitution and the TATA-box replacement was about 18-fold more active than the wt gene in the in vitro transcription system. These results suggest that the in vitro system may preferentially build a functional transcription complex with components that do not normally interact with the wt rpS16 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The developmentally regulated shift from membrane to secreted mu mRNA production is accompanied by an increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency.

To determine whether there are any developmental changes in the efficiencies of cleavage-polyadenylation or splicing reactions that could affect the usage of weak (suboptimal) processing signals and thus provide a basis for the regulated production of mu m versus mu s mRNA during B-lymphocyte maturation, we studied the expression of transfected mu genes in which the natural competition between cleavage-polyadenylation and splicing was replaced by alternative usage of tandem weak and strong poly(A) sites or by competition between suboptimal and optimal 5' splice junctions. Our results indicate that there is a 50 to 100% increase in cleavage-polyadenylation efficiency but no measurable change in splicing efficiency as maturation proceeds from the B-cell to plasma cell stage.

Adenovirus Early Proteins↗
PubMed1991

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Communicable Disease Control↗
PubMed1990

The temporal order of appearance of transcripts from unrearranged and rearranged Ig genes in murine fetal liver.

The developmental time course of RNA transcribed from unrearranged (germ-line) and rearranged Ig genes in murine fetal liver was determined by a quantitative Northern blot analysis. Sterile Cmu transcripts and germ-line VH transcripts are detectable as early as day 14, whereas significant amounts of rearranged VDJCmu H chain transcripts do not appear until days 16 to 17. The sterile Cmu transcripts continuously increase in abundance throughout fetal development, in contrast to the germ-line VH transcripts, which decrease abruptly after day 16. Transcripts of germ-line and rearranged CK genes are detectable on day 17, and continue to increase in abundance on day 18. Transcripts from a pre-B cell specific gene, lambda 5, first appear on day 15, and reach maximum abundance on day 17. The order of these events is consistent with the known order of gene rearrangements and with the idea that transcriptional activation of germ-line loci is a prerequisite for Ig gene rearrangement. The lag between the onsets of germ-line Cmu and VH transcription and the appearance of VDJCmu transcripts suggests that additional developmentally regulated events may be necessary to achieve efficient expression of completely rearranged H chain genes.

Animals↗
PubMed1990

A novel upstream element compensates for an ineffectual octamer motif in an immunoglobulin V kappa promoter.

The octamer (or dc/cd) motif is considered to be a critical component of all immunoglobulin (Ig) promoters. Although the sequence of this motif is highly conserved among most Ig promoters, there are some notable examples in which efficiently expressed Ig genes contain divergent octamers with base substitutions that are demonstrably deleterious when tested with heterologous proximal promoter elements. To elucidate the mechanisms that enable these naturally occurring Ig genes to cope with divergent octamers, we analyzed two such promoters with regard to their ability to interact with relevant transcription factors. We found that the divergent octamer in the kappa O germline promoter strongly binds both Oct-1 and Oct-2 factors, presumably because of compensatory contributions by flanking DNA sequences. A more surprising result was obtained with the V kappa 19 promoter. In this case, the divergent octamer is a very weak Oct factor binding site and, without help from another upstream element, is inadequate for efficient promoter function. This additional element, termed kappa Y because of its high pyrimidine content (CTTCCTTA), serves as a binding site for a novel lymphoid-specific factor. When the divergent V kappa 19 octamer was converted to a strong Oct factor binding site by a single point mutation, the need for kappa Y was obviated. Interestingly, VH promoters that contain the same divergent octamer also contain an upstream element that is very similar to kappa Y.

Animals↗
PubMed1990

Functional dissection of a mouse ribosomal protein promoter: significance of the polypyrimidine initiator and an element in the TATA-box region.

All of the mammalian ribosomal protein (rp) genes examined to date initiate transcription with high precision despite the fact that they do not contain a well-defined TATA box. The initiation sites are situated within polypyrimidine tracts that are flanked by both upstream and intragenic promoter elements. In the TATA-box region of each rp promoter, there is a functionally critical element with nuclear factor binding specificity that is distinct from that of a conventional TATA box. To understand how the various elements contribute to rp promoter function, we have used site-specific mutagenesis-transfection protocols and factor binding analyses to evaluate the significance of the polypyrimidine initiator and the TATA-box counterpart for efficient and accurate transcription of the rpS16 gene. Our results indicate (i) that the polypyrimidine initiator sequence critically defines the position of the transcriptional start site, whereas a much less specific sequence is sufficient to satisfy the efficiency requirement; (ii) that an uninterrupted stretch of pyrimidines in the initiator region is not necessary for efficient transcription of rpS16 gene; and (iii) that the TATA-box counterpart or even a substituted conventional TATA box primarily influences promoter efficiency. The great diversity of promoter design, which is becoming evident as more RNA polymerase II promoters are being carefully dissected, suggests that the requirements for building a functional initiation complex may be much more flexible than was previously appreciated.

Animals↗
PubMed1990

Translational control of ribosomal protein production in mammalian cells.

Mammalian ribosomal protein (rp) mRNAs are subject to translational control, as illustrated by their selective release from polyribosomes in growth-arrested cells and their under-representation in polyribosomes of normally growing cells. Recent studies have localized the translational regulatory element to the 5' end of the rp mRNA and have demonstrated that an oligopyrimidine tract, which adjoins the cap structure in all known vertebrate rp mRNAs, is an essential part of this element. Possible factors that might interact with the oligopyrimidine tract are discussed.

Animals↗
PubMed1989

A characterization of the elements comprising the promoter of the mouse ribosomal protein gene RPS16.

The elements comprising the mouse rpS16 promoter were characterized by transfection experiments with mutant genes in which various portions of the 5' flanking region and exon I were removed or substituted with extraneous DNA sequence. These experiments were carried out with otherwise intact rpS16 genes transfected into monkey kidney (COS) cells and also with chimeric rpS16-CAT gene constructs transfected into mouse plasmacytoma cells and COS cells. The locations of the functionally important elements were generally correlated with the locations of binding sites for specific nuclear factors, which were identified by gel-mobility shift analyses and methylation interference footprints. The most upstream element, which is located approximately 165 bp from the cap site, binds the Sp1 transcription factor and augments the promoter activity by 2 to 2.5-fold. In addition, there is a complex bipartite element in the -83 to -59 region, an element in the -37 to -12 region and an element in the +9 to +29 region of exon I, all of which are essential for rpS16 expression. The rpS16 promoter has a general architecture that resembles other mouse rp promoters; however, it also possesses some distinctive characteristics.

Animals↗
PubMed1989

An element downstream of the cap site is required for transcription of the gene encoding mouse ribosomal protein L32.

To identify the elements that regulate transcription of the mouse gene encoding ribosomal protein L32 (rpL32), we transfected monkey kidney (COS or CV-1) cells with mutants bearing progressive 5' deletions or an internal deletion in exon I and measured their transient expression by S1 nuclease protection analysis. When the mutant genes were tested in the vector pi SVHSplac, which contains a short segment of the oriregion of simian virus 40, maximum expression was observed with as little as 36 base pairs of 5' flanking sequence, and the mutant bearing the exon I deletion was expressed very efficiently. However, when the genes were tested in a simple prokaryotic (pUC) vector, the expression was increased 3- to 4-fold by sequences between -36 and -159, and the exon I segment was absolutely required for expression. Gel mobility-shift and methylation interference analyses revealed that a nuclear factor specifically binds to a GGCTGCCATC sequence within this exon I segment. These results, taken together with other recent findings, indicate that the elements involved in transcriptional regulation of the rpL32 gene are distributed over a 200-base-pair region that spans the cap site. The contributions of some of these elements are apparently masked in the presence of simian virus 40 ori-region elements.

Animals↗
PubMed1989

Equipotent mouse ribosomal protein promoters have a similar architecture that includes internal sequence elements.

The promoters of the mouse ribosomal protein genes rpL30, rpL32, and rpS16 are of equal strength, as indicated by in vivo measurements of polymerase loading and by their relative efficiency in driving the expression of a linked reporter gene. The equipotency of these promoters appears to derive from a remarkably similar architecture in which five or more elements are distributed over a 200-bp region that spans a polypyrimidine-embedded cap site. Three trans-acting factors are shared by the rpL30 and rpL32 promoters, one of which, delta, recognizes a common CNGCCATCT motif in the first (untranslated) exons. Site-specific mutagenesis demonstrated that delta-factor binding is critical for rpL30 promoter function. The repeated occurrence of this novel promoter architecture among ribosomal protein genes with very different coding specificities is most readily explained by convergent evolution.

Animals↗
PubMed1989

The regulated production of mu m and mu s mRNA is dependent on the relative efficiencies of mu s poly(A) site usage and the c mu 4-to-M1 splice.

The relative abundance of the mRNAs encoding the membrane (mu m) and secreted (mu s) forms of immunoglobulin mu heavy chain is regulated during B-cell maturation by a change in the mode of RNA processing. Current models to explain this regulation involve either competition between cleavage-polyadenylation at the proximal (mu s) poly(A) site and cleavage-polyadenylation at the distal (mu m) poly(A) site [poly(A) site model] or competition between cleavage-polyadenylation at the mu s poly(A) site and splicing of the C mu 4 and M1 exons, which eliminates the mu s site (mu s site-splice model). To test certain predictions of these models and to determine whether there is a unique structural feature of the mu s poly(A) site that is essential for regulation, we constructed modified mu genes in which the mu s or mu m poly(A) site was replaced by other poly(A) sites and then studied the transient expression of these genes in cells representative of both early- and late-stage lymphocytes. Substitutions at the mu s site dramatically altered the relative usage of this site and caused corresponding reciprocal changes in the usage of the mu m site. Despite these changes, use of the proximal site was still usually higher in plasmacytomas than in pre-B cells, indicating that regulation does not depend on a unique feature of the mu s poly(A) site. Replacement of the distal (mu m) site had no detectable effect on the usage of the mu s site in either plasmacytomas or pre-B cells. These findings are inconsistent with the poly(A) site model. In addition, we noted that in a wide variety of organisms, the sequence at the 5' splice junction of the C mu 4-to-M1 intron is significantly different from the consensus 5' splice junction sequence and is therefore suboptimal with respect to its complementary base pairing with U1 small nuclear RNA. When we mutated this suboptimal sequence into the consensus sequence, the mu mRNA production in plasmacytoma cells was shifted from predominantly mu s to exclusively mu m. This result unequivocally demonstrated that splicing of the C mu 4-to-M1 exon is in competition with usage of the mu s poly(A) site. A key feature of this regulatory phenomenon appears to be the appropriately balanced efficiencies of these two processing reactions. Consistent with predictions of the mu s site-splice model, B cells were found to contain mu m precursor RNA that had undergone the C mu 4-to-M1 splice but had not yet been polyadenylated at the mu m site.

Animals↗
PubMed1989

Localization of transcriptional regulatory elements and nuclear factor binding sites in mouse ribosomal protein gene rpL32.

The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150- to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to identify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 in exon I, and within the first 31 base pairs of intron 1. One of these factors may bind to multiple sites within the promoter region. Interestingly, the factor that binds to a sequence motif in the first exon also binds to similar motifs in a comparable region of the c-myc gene.

Animals↗
PubMed1989

Importance of introns for expression of mouse ribosomal protein gene rpL32.

The importance of intronic sequences for expression of the mouse ribosomal protein gene rpL32 was evaluated by transfection experiments with a series of mutant constructs in which one or more of the three rpL32 introns was totally or partially deleted. When transiently transfected into monkey kidney (COS) cells or stably transfected into mouse L cells, a mutant that lacked all three introns was completely inactive. Constructs that contained intron 1, either alone or in combination with another intron, were expressed as efficiently as was the normal intact rpL32 gene. Constructs that lacked intron 1 but contained another spliceable intron, even one from a foreign gene, were expressed at about 10 to 20% of the maximum level. These results indicated that intron 1 contains an element that increases the level of expression by 5- to 10-fold. A comparison of internal deletion mutants localized the element to within the first 27 base pairs of intron 1. Nuclear run-on experiments with stably transfected COS cells demonstrated that this element functions at the transcriptional level. The element was inactive when translocated to a position upstream of the transcriptional start site or to a position within intron 3, which indicated that it does not have the properties of a typical enhancer. From these and other results, we conclude that introns have both a general and a specific role in rpL32 expression. The general role, which can be satisfied by any spliceable intron, is to ensure an efficient yield of RNA transcripts. The specific role is uniquely attributable to intron 1, which contains a transcriptional regulatory element near its 5' end.

Animals↗
PubMed1988

Complementation between two cell lines lacking kappa enhancer activity: implications for the developmental control of immunoglobulin transcription.

Plasmacytoma S107 and the pre-B cell line 3-1 both lack immunoglobulin kappa (Ig kappa) enhancer activity due to the absence of the active form of a trans-acting nuclear factor, NF-kappa B, which binds to and activates the kappa enhancer. Pre-B cells possess the factor in a masked form and can activate it by a post-translational mechanism after treatment with specific inducing agents. In the experiments presented here somatic cell hybrids were used to determine whether S107 cells also possess NF-kappa B in a masked form, or alternatively, whether they possess the activation system but lack the factor. We observed that hybrids between S107 and pre-B cells produce the active form of NF-kappa B and exhibit transcriptional activation of previously silent kappa loci. These results demonstrate that S107 cells totally lack factor NF-kappa B but not the ability to activate it. Treatment of the hybrid cells with bacterial lipopolysaccharide (LPS), increases the NF-kappa B titer 4- to 5-fold and causes a concomitant 4- to 5-fold increase in kappa enhancer activity within these cells. However, the expression of the activated kappa loci remains unchanged after LPS-treatment, indicating that they are no longer under the control of the kappa enhancer. Therefore, a two-step transcriptional process occurs in these cells. First, the silent kappa loci are activated by the production of factor NF-kappa B. Subsequently, a second transcriptional mechanism overrides the dependence on the kappa enhancer and maintains kappa transcription at a constant level regardless of the level of kappa enhancer activity within the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗