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T R Broker

Publications and source records attributed to T R Broker.

At least 73 records · Page 4Linked to original sources
PubMed1987

Identification and mapping of human papillomavirus type 1 RNA transcripts recovered from plantar warts and infected epithelial cell cultures.

Multiple spliced transcripts of human papillomavirus type 1 were detected by electron microscopic analysis of R-loops formed with total RNA extracted from plantar warts and with poly(A)+ RNA isolated from cultured keratinocytes infected with human papillomavirus type 1. The 5' ends of the RNAs were mapped to sites in the E7 open reading frame (ORF), just upstream of the E6 ORF and in the upstream regulatory region. Species with 5' ends in E7 accounted for over 95% of all transcripts seen. Two polyadenylation sites were used, one at the end of the early (E) region of the viral DNA, the other at the end of the late (L) region. The most abundant species had a short 5' exon of approximately 100 nucleotides spanning the junction of the E7 and E1 ORFs spliced to a 3' exon of 800 nucleotides in the region with overlapping E2 and E4 ORFs; it was polyadenylated at the end of the E region. This species probably encodes the abundant E4 protein found in plantar warts (F. Breitburd, O. Croissant, and G. Orth, Cancer Cells, vol. 5, in press; J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986). Other transcripts had exons spanning the E6-E7 ORFs, the E4-E5-L2-L1 ORFs, or the L1 ORF. The infrequent L1 transcript, probably the mRNA coding for the major capsid protein, had the same 5' exon in E7 as the abundant mRNA spliced from E1 and E4 ORFs, suggesting genetic regulation via the choice of the alternative polyadenylation sites or mRNA processing.

Base Sequence↗
PubMed1987

Human papillomavirus types 6 and 11 mRNAs from genital condylomata acuminata.

We have identified and mapped a number of RNA species of human papillomavirus types 6 and 11 from condylomata acuminata by the electron microscopic R-loop technique. Each of the early (E)- and late (L)-region open reading frames (ORFs) deduced from the DNA sequences was represented in one or more transcripts. In addition, RNA species that could encode the modulator of DNA replication and the repressor of transcription, functions recently identified in the genetically similar bovine papillomavirus type 1, were also detected. Some ORFs were 5' proximal in one or more transcripts, whereas others were not 5' proximal in any species, suggesting that internal initiation of translation might be required to gain access to these latter ORFs. Virtually all transcripts had their 5' ends located in the E region and were polyadenylated at one of two sites, i.e., at the end of the E region or at the end of the L region. The great majority of the RNAs were derived from the E region of the genome, with one species approximately 50 to 100 times more abundant than the others. For most of the RNAs, the 5' end mapped near nucleotide 700; minor populations had 5' ends near nucleotide 100 or 1200. By correlating our mapping data with the genomic DNA sequences as well as available RNA structures and cDNA sequences of several papillomaviruses, we predict a number of mRNA splice donor and acceptor sites and suggest that the papillomaviruses have sophisticated usage of ORFs through alternative promoters, mRNA splice sites, and polyadenylation sites.

Anus Neoplasms↗
PubMed1987

Enhancers and trans-acting E2 transcriptional factors of papillomaviruses.

The upstream regulatory regions of human papillomavirus (HPV) types 1, 6b, 7, 11, 16, and 18, bovine papillomavirus type 1, and cottontail rabbit papillomavirus were cloned into transcriptional enhancer assay plasmids which carry the simian virus 40 early promoter lacking its own enhancer and the bacterial gene encoding chloramphenicol acetyltransferase (EC 2.3.1.28) (CAT). Enhancer activity, reflected by CAT gene expression, was detected in all of the upstream regulatory regions tested only when the recombinants were cotransfected with plasmids which express an intact E2 open reading frame of HPV types 1 and 11 or bovine papillomavirus type 1. Each E2 protein stimulated the enhancer from the same virus and, to somewhat lesser degrees, also those from the heterologous viruses. Hence, the enhancer and the E2 protein are functionally conserved among papillomaviruses. There was some nonreciprocity in the extent of trans-activation in heterologous E2-enhancer interactions. Primer extension analyses demonstrated that the E2 proteins increased the abundance of CAT gene mRNA. Tandem multiplication of the HPV type 11 enhancer sequence dramatically increased its response to E2 stimulation; this is possibly relevant to the pathogenicity of papillomaviruses.

Cloning, Molecular↗
PubMed1987

Structure and genetic expression of papillomaviruses.

This article reviews the physical structures, genetic organization, expression, and regulation of papillomaviruses. In view of the extensive characterization of cellular transformation and viral functions that have been achieved in experimentally infected animals and in transformed cell cultures, the bovine papillomavirus type 1 (BPV-1) and Shope cottontail rabbit papillomavirus (CRPV) are compared and contrasted with the human papillomaviruses.

Animals↗
PubMed1986

In situ hybridization detection of human papillomavirus DNAs and messenger RNAs in genital condylomas and a cervical carcinoma.

Routinely processed formalin-fixed paraffin-embedded sections from anogenital condyloma acuminatum and an invasive squamous cell carcinoma of the cervix were examined by in situ hybridization for the detection of human papillomavirus (HPV) DNAs and messenger RNAs. Asymmetric, single-stranded, tritium-labeled RNA probes for both the coding and the nonsense strands of HPVs 6, 11, 16, 18, and 31 were hybridized and washed under stringent conditions and detected by autoradiography. Type-specific HPV DNAs were detected with specific nuclear localization, while HPV messenger RNAs gave much higher signals and had clear-cut cytoplasmic localization. Cross-hybridization was observed only with closely related viruses. The level of signal obtained seemed to be linked to the degree of cellular differentiation, with koilocytotic cells labeling the most heavily. However, messenger RNA could be detected in even relatively undifferentiated cells within areas of dysplasia and invasive carcinoma. In situ hybridization is a sensitive and specific method for investigation of the dynamic interplay of papillomavirus replication and gene expression, cellular differentiation, and neoplastic transformation.

Carcinoma, Squamous Cell↗
PubMed1981

Sequences of human adenovirus Ad3 and Ad7 DNAs encoding the promoter and first leader segment of late RNAs.

DNA segments containing the major promoter at coordinate 16.5 for rightward transcription from human adenovirus serotypes 3 and 7 (Ad3 and Ad7), two closely related class B viruses, have been sequenced and found virtually identical. Furthermore, over 80% of the nucleotides of Ad3 and Ad7 in this entire region are homologous to their counterparts in the DNA of the more distantly related class C serotype Ad2. There are the same number of nucleotide pairs among these serotypes within the region compared. Most changes are transitions or transversions and the several single-base deletions are always compensated by nearby insertions. These few changes nonetheless result in 24 differences between Ad7 (or Ad3) and Ad2 in a total of 32 cleavage sites. The promoter for the rightward-transcribed RNAs and the first segment of the consanguinous tripartite leader found at the 5'-ends of all the later mRNAs derived from that promoter have been identified by analogy to the nucleotide sequences of Ad2. In particular, the "Hogness box" or RNA polymerase staging site for the major rightward transcription unit is completely homologous to that of Ad2. There are only six bp changes in the first late leader segment despite previous evidence suggesting that they might be quite heterologous. A prominent dyad axis of symmetry exists just upstream from the presumed 5'-end of the late RNA. However, unlike the stem-loop structure proposed for Ad2 by Ziff and Evans (1978), the base changes relative to Ad2 mandate a different potential stemloop structure in the single strand of Ad3 and Ad7 DNAs. This hairpin places the "Hogness box" immediately next to the 5'-end of the RNA at the base of stem. An analogous dyad axis of symmetry or stemloop structure can be found in a number of eukaryotic systems, including the major rightward transcription unit of Ad2. This feature may be of relevance to the positioning of RNA polymerase II on the DNA and to the promotion of transcription.

Adenoviruses, Human↗
PubMed1979

Comparison of late mRNA splicing among class B and class C adenoviruses.

Adenovirus class B (Ad3 and Ad7) and class C (Ad1, Ad5, and Ad6) late r-strand mRNA's were found to have segmented 5' leaders. These leaders were very similar among serotype within a class but differed in sequence from the leaders on late mRNA's of a different class. However, the leader components of class B viruses mapped at essentially the same map coordinates as those of class C viruses. The 5' coordinates of the main bodies of class B messages to which the tripartite leaders are attached as well as the map positions of several of their early mRNA's were very similar to those of Ad2 transcripts. Infrequent examples of late r-strand polysomal RNAs of Ad3 and Ad7 had, in addition to the three common leader segments, a fourth leader segment derived from RNA encoded at various sites between the second and third leaders. The extra components formed several distinct groups. These molecules are presumably intermediates in the splicing processes that generate mature messages.

Adenoviruses, Human↗
PubMed1978

The spliced structures of adenovirus 2 fiber message and the other late mRNAs.

All late adenovirus 2 messenger RNAs, except perhaps that for peptide IX, are composite molecules with sequences derived from at least two to five or more separate portions of the genome, as determined by electron microscopic analyses of polysomal RNA hybridized to single- or double-stranded DNA. We previously reported that eight different rightward-transcribed mRNAs contain, at their 5' ends, a common three-part leader sequence derived from coordinates 16.6, 19.6 and 26.6, and that a ninth mRNA consists of sequences from 4.6--6.1 linked directly to those from 9.7--11.0 (Chow et al., 1977a; revised coordinates). We now demonstrate that four additional RNA species also have the same tripartite leader joined to messages with 5' ends located at coordinates at 29.1, 30.5, 33.9 and 42.8. The RNA transcript extending from 29.1--39.0 covers most or all of the viral associated (VA) RNA genes (29.0--30.1). The late RNAs can be grouped into several families of transcripts in which two or three mRNAs have the same 3' end and 3' proximal sequences, but differ substantially in length at the 5' end and, therefore, the coordinate at which the leader sequences are attached to the main body of the message. The number of RNA species transcribed from the Ad2 chromosome between coordinates 29 and 50 exceeds the number of identified Ad2 proteins. Several pairs of messages differing in length by a few percent may thus encode the same protein or have a precursor-product relationship. We have also found that a subpopulation of the polysomal mRNAs for the fiber protein, the most distal gene (86.3--91.2) in the rightward transcription unit, has a fourth leader component "y" (78.6--79.1) in addition to the common tripartite leader sequences. Less frequent fiber RNA species have fifth "x" (76.9--77.3) and/or sixth "z" (84.7--85.1) leader segments. Transcripts with x, y or z segments might be processing intermediates, and it is possible to arrange these species into an array suggestive of multiple pathways for the maturation of fiber mRNA. mRNA transcribed at late times from the L strand between coordinates 15.0 and 11.0 has a single component leader located 200 bases from the main body of the gene from coordinate 16.1 to 15.7. This leader originates from a site very close to the first leader component (16.5--16.6) of the R strand messages and suggests the presence of adjacent L and R strand promoters from which transcription diverges. Taken together, these results emphasize the diversity of splicing patterns and the variety of recombined sequences generated during the synthesis of late Ad2 mRNAs.

Adenoviruses, Human↗
PubMed1978

Electron microscopic visualization of tRNA genes with ferritin-avidin: biotin labels.

A method is described for indirect electron microscopic visualization and mapping of tRNA and other short transcripts hybridized to DNA. This method depends upon the attachment of the electron-dense protein ferritin to the RNA, the binding being mediated by the remarkably strong association of the egg white protein avidin with biotin. Biotin is covalently attached to the 3' end of tRNA using an NH2(CH2)5NH2 bridge. The tRNA-biotin adduct is hybridized to complementary DNA sequences present in a single stranded non-homology loop of a DNA:DNA heteroduplex. Avidin, covalently crosslinked to ferritin, is mixed with the heteroduplex and becomes bound to the hybridized tRNA-biotin. Observation of the DNA:RNA-biotin:avidin-ferritin complex by electron microscopy specifically and accurately reveals the position of the tRNA gene, with a frequency of labeling of approximately 50%.

Avidin↗
PubMed1978

Adjacent insertion sequences IS2 and IS5 in bacteriophage Mu mutants and an IS5 in a lambda darg bacteriophage.

Using electron microscopic heteroduplex analysis, we have demonstrated that an insertion found in a Mu prophage and in some infectious. Mu deletion-substitution mutants derived from it consists of bacterial insertion sequence IS2 linked directly to IS5. Other infectious Mu mutants derived from the same lysogen have only IS5 or a portion of IS2. In addition, we have found that an independent insertion in a transducing phage, lambda 13 dargB2, is IS5. The ends of IS5 are short, inverted duplications of each other. These observations support the notion that the DNA insertion previously designated IS5 on the basis of a single example in lambda KH100 is a bona fide bacterial insertion sequence.

Coliphages↗