Biomedical subjects
Publications and source records attributed to R A Owens.
Surgeons perform total shoulder arthroplasty (TSA) procedures to reduce patients' intractable arthritic pain and to repair humeral head fractures. Total shoulder arthroplasty has undergone remarkable advances--not only in prosthetic improvements and refinements--but in patient outcomes. Advantages to TSA procedures include decreased pain and patients' increased ability to perform activities of daily living.
The Rep proteins of adeno-associated virus type 2 (AAV) are known to bind to Rep recognition sequences (RRSs) in the AAV inverted terminal repeats (ITRs), the AAV p5 promoter, and the preferred AAV integration site in human chromosome 19, called AAVS1. Integration of the AAV genome into AAVS1 appears to be mediated by an interaction between the Rep proteins of AAV and Rep binding sites within the viral genome and the integration locus. In an attempt to identify potential alternate integration sites, we looked for recognition sites for AAV Rep proteins in the human genome by performing a BLASTN computerized homology search. We used the 16-mer core sequences of the RRSs in the AAV ITRs and AAVS1 separately as query sequences and identified 18 new RRSs in or flanking the genes coding for the following: tyrosine kinase activator protein 1 (TKA-1); colony stimulating factor-1; insulin-like growth factor binding protein 2 (IGFBP-2); histone H2B.1; basement membrane heparan sulfate proteoglycan, also known as perlecan; the AF-9 gene product, which is involved in the chromosomal translocation t (9:11)(p22:q23); the betaB subunit of the hormone known as inhibin; interleukin-2 enhancer binding factor; an endoplasmic reticulum-Golgi intermediate compartment resident protein called p63; a global transcription activator (hSNF2L); the beta-actin repair domain; a retinoic acid-inducible factor, also known as midkine; a breast tumor autoantigen; a growth-arrest- and DNA-damage-inducible protein called gadd45; the cyclin-dependent kinase inhibitor called KIP2, which inhibits several G1 cyclin-cyclin-dependent kinase complexes; and the hereditary breast and ovarian cancer gene (BRCA1). RRSs were also identified in a newly discovered open reading frame on chromosome 10 and in the ERCC1 locus on human chromosome 19. The ability of a maltose binding protein-Rep68 fusion protein to bind to these sequences was confirmed by electrophoretic mobility shift assays. These sites may serve as alternate integration sites for AAV or play a role in Rep-mediated effects on human cells.
The Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which contain overlapping amino acid sequences. They are required for viral replication and preferential integration of the AAV genome into a region of human chromosome 19. During the terminal resolution process of AAV DNA replication, these proteins make a site-specific and strand-specific endonuclease cut within the AAV inverted terminal repeat DNA. The Rep68 and Rep78 proteins also have helicase and DNA-binding activities. The endonuclease activity is believed to involve the covalent attachment of Rep68 or Rep78 at the cut site via a phosphotyrosine linkage. In an attempt to identify the active-site tyrosine residue of Rep78 and Rep68, tyrosine residues were site specifically mutated to phenylalanines by overlap extension PCR, and the resulting PCR fragments were cloned into a maltose binding protein-Rep68 fusion (MBP-Rep68delta) expression vector. The mutant MBP-Rep68delta proteins were expressed in Escherichia coli cells, purified with amylose resin, and assayed in vitro for Rep68-specific activities. Although several of the mutations disrupted the endonuclease activity, only the mutation of tyrosine 152 abrogated the endonuclease activity with no discernible effect on the helicase or DNA-binding activities. Our data therefore suggest that there are distinct active sites for the helicase and endonuclease activities.
The native structure of potato spindle tuber viroid (PSTVd) contains a series of short double helices and small internal loops that are organized into five structural domains. Nucleotides within the pathogenicity domain are known to play a critical role in modulating PSTVd symptom expression, and it has been suggested that disruption of a comparatively unstable "premelting region" within the pathogenicity domain may be required for disease induction. We have used a combination of quantitative bioassays, temperature gradient gel electrophoresis of circularized RNA transcripts, and thermodynamic calculations to compare the biological and structural properties of 12 representative PSTVd sequence variants. Certain mutations appeared to act indirectly, downregulating pathogenicity by suppressing the rate of PSTVd replication/accumulation. The effects of other mutations appeared to be more direct, but there was no consistent correlation between symptom severity and melting temperature. Taking into account the three-dimensional shape of RNA helices, comparison of the optimal secondary structures for these variants point to major differences in the geometry of their pathogenicity domains; i.e., variants producing intermediate symptoms possess a linear arrangement of three consecutive helices, whereas for variants producing mild or severe symptoms this domain is bent in opposing directions. Such alterations in RNA structure together with concomitant alterations in RNA-protein interaction(s) may be the primary cause of viroid pathogenicity.
Potato spindle tuber viroid (PSTVd) is believed to undergo a series of specific structural transitions during replication. The variable domain of PSTVd is known to contain sequences that are important for replication/accumulation as well as one of three "premelting regions" which control breakdown of the native structure in vitro. We have examined the structural and biological effects of five single and two double nucleotide substitutions within premelting region 3 in an effort to isolate temperature-sensitive mutations affecting PSTVd replication or pathogenesis. None of these mutants replicated as rapidly as the wild type, and a variety of spontaneous sequence changes were detected in their progeny. Higher temperatures were able to partially overcome the inhibition of replication associated with a more stable secondary structure, but no well-defined temperature-sensitive PSTVd mutants were identified. Selective pressures arising from the interaction of assay temperature and structural stability in vivo appear capable of moving PSTVd populations between peaks of relatively high fitness. Depending on the exact nature and location of the mutation, selection may occur at the level of either the plus or the minus strand.
Adeno-associated virus type 2 (AAV) Rep proteins have been postulated to play a role in unwinding the 145-bp inverted terminal repeats during AAV DNA replication. Previous studies showed that AAV Rep78 and Rep68 could unwind a DNA partial duplex of 26 bp. In this work it is demonstrated that nuclear extracts of human 293 cells containing wild-type Rep68 can unwind partial DNA duplexes up to 160 bp long. Mutant Rep proteins with either a histidine substituted for lysine 340 or a deletion of methionine 225 had no detectable helicase activity and inhibited the helicase activity of wild-type Rep68 protein. This observation is consistent with the model that the functional form of the Rep proteins is a multimer.
The effect of the temporal spacing and degree of milk removal by either expression or breastfeed on short-term (hour to hour) rates of milk synthesis was investigated. For four expressing mothers (three of whom had given birth prematurely), the relationship between milk produced at an expression and the time since the last expression showed that proportionally less milk is produced after intervals of 6-18 h than after intervals of less than 6 h (P < 0.0002). For five breastfeeding and four expressing mothers (three of whom had given birth prematurely), rates of milk synthesis (ranging from 0 to 56 ml/h) were linear between breastfeeds and expressions (intervals ranging from < 1 h to 6 h). Changes in rate of milk synthesis were not directly associated with the frequency of breastfeeds but rather with the degree of emptying of the breast (P < 0.05). These results indicate the importance of degree, rather than frequency, of breast emptying in the short-term control of human milk synthesis.
In women, the concentration of prolactin in the plasma increases in response to nipple stimulation. This response has led to the assumption that prolactin influences the rate of milk synthesis. To investigate this hypothesis we have measured 24 h milk production, the short-term (between breastfeeds) rates of milk synthesis and the concentration of prolactin in the blood and breastmilk, from 1 to 6 months of lactation in eleven women. Over the long term, the 24 h milk production remained constant (means +/- S.E.M.): 708 +/- 54.7 g/24 h (n = 11) and 742 +/- 79.4 g/24 h (n = 9) at 1 and 6 months, respectively. The average short-term rate of milk synthesis (calculated from the increase in breast volume between breastfeeds; means +/- S.E.M.) did not change: 23 +/- 3.5 ml/h (n = 23) and 23 +/- 3.4 ml/h (n = 21) at 1 and 6 months, respectively. However, significant variation in the short-term rate of milk synthesis (from < 5.8 to 90 ml/h) was found both between breasts, measured concurrently (coefficient of variation, c.v. = 72%), and within the same breast, measured over consecutive breastfeeds (c.v. = 85%). The basal and suckling-stimulated concentrations of prolactin in the plasma (means +/- S.E.M.) declined from 1 to 6 months (basal, from 119 +/- 93 to 59 +/- 29 micrograms/1; peak, from 286 +/- 109 to 91 +/- 44 micrograms/l). In contrast, the concentration of prolactin in milk was much lower than in plasma, and decreased only slightly from 1 to 6 months of lactation (fore-milk, from 26.4 +/- 10 to 23.3 +/- 9.8 micrograms/l; hind-milk, from 18.9 +/- 5.1 to 13.2 +/- 6.3 micrograms/l). The concentration of prolactin in the milk was related to the degree of fullness of the breast, such that the concentration was highest when the breast was full. We found no relationship between the concentration of prolactin in the plasma and the rate of milk synthesis in either the short or long term. However, the relationship between the concentration of prolactin in milk and the degree of fullness of the breast suggests that the internalization of prolactin, after binding to its receptor, may be restricted when the alveolus is distended with milk.
Both the Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) bind to AAV terminal repeat hairpin DNA and can mediate site-specific nicking in vitro at the terminal resolution site (trs) within the terminal repeats. To define the regions of the Rep proteins required for these functions, a series of truncated Rep78 derivatives was created. Wild-type and mutant proteins were synthesized by in vitro translation and analyzed for AAV hairpin DNA binding, trs endonuclease activity, and interaction on hairpin DNA. Amino-terminal deletion mutants which lacked the first 29 or 79 amino acid residues of Rep78 did not bind hairpin DNA, which is consistent with our previous identification of a DNA-binding domain in this region. Progressive truncation of the carboxyl-terminal region of Rep78 did not eliminate hairpin DNA binding until the deletion reached amino acid 443. The electrophoretic mobility of the Rep-specific protein-DNA complexes was inversely related to the molecular weight of the Rep derivative. Analysis of the C-terminal deletion mutants by the trs endonuclease assay identified a region (amino acids 467 to 476) that is essential for nicking but is not necessary for DNA binding. When endonuclease-positive, truncated Rep proteins that bound hairpin DNA were mixed with full-length Rep78 or Rep68 protein in electrophoretic mobility shift assays, a smear of protein-DNA complexes was observed. This smear migrated at an intermediate position with respect to the bands generated by the proteins individually. An antibody recognizing only the full-length protein produced a novel supershift band when included in a mixed binding assay containing Rep68 and a truncated Rep mutant. These experiments suggest that the Rep proteins can form hetero-oligomers on the AAV hairpin DNA.
Rep68 protein, encoded by adeno-associated virus type 2 (AAV), has been previously shown to bind to specific sequences within the viral genome and in human chromosome 19. The effect of AAV Rep protein on human cellular genes is of interest because AAV is being developed as a gene therapy vector. We have identified sequences related to the Rep recognition sequence in the AAV P5 promoter in or near the c-sis proto-oncogene and the genes coding for a hepatocyte glucose transporter, alpha-A-crystallin, and carcinoma marker GA733-1. The ability of Rep68 to bind to these sites was established by gel shift assays, and the effect of Rep68 on the expression of these genes was tested by semiquantitative reverse transcriptase PCR. Rep68 enhances the expression of the c-sis proto-oncogene, which codes for the B polypeptide of platelet-derived growth factor, a multifunctional growth factor that is involved in embryonic development, tissue regeneration, osteogenesis, fibrosis, atherosclerosis, and neoplasia.
Nucleotides within the pathogenicity domain of potato spindle tuber viroid (PSTVd) are known to play an important role in regulating symptom expression, but the underlying molecular mechanism is unknown. In order to determine more precisely how structural features within the pathogenicity domain regulate symptom expression, we have characterized a series of mutations that progressively stabilize premelting region 1 and the rest of the "virulence modulating" region. The structural effects of these mutations were monitored by temperature gradient gel electrophoresis of circularized RNA transcripts, and their biological effects were assessed by quantitative bioassays in tomato. Closure of a 4-nucleotide loop within the premelting region 1 virtually abolished PSTVd infectivity, especially when a nearby 2-nucleotide loop was also closed. Although RNA transcripts containing less stabilizing mutations were readily infectious, none of the four single and one double substitutions examined were stably maintained in vivo. The pattern of spontaneous, apparently compensatory sequence changes observed in the progeny suggests that PSTVd variants with less stable secondary structures enjoy a selective advantage. Mutations which stabilize the pathogenicity domain of PSTVd in vitro also suppressed symptom expression, but at least one other mutation having no obvious structural effects was associated with a similar phenotype. Conformational stability appears to be only one of several factors regulating PSTVd replication and pathogenicity.
The 303 nucleotide sequence of HSVd-citrus(T), a hop stunt viroid (HSVd) variant present in Etrog citron growing in Taiwan, was determined from cDNAs amplified by the polymerase chain reaction. HSVd-citrus(T) is very similar to several HSVd isolates previously recovered from citrus or cucumber, and exhibits microsequence heterogeneity at positions 154 and 181. Phylogenetic analysis using maximum parsimony grouped HSVd-citrus(T) with seven other isolates from citrus and cucumber in a large cluster of "citrus-type" isolates. A similar analysis revealed marked differences in both the extent and distribution of sequence variation among naturally occurring isolates of potato spindle tuber viroid.
Transcript levels from the P5 promoter of adeno-associated virus type 2 (AAV) are negatively regulated by the AAV Rep78 and Rep68 proteins in the absence of helper virus. We have identified a Rep-responsive negative cis element of the P5 promoter between the P5 TATA box and transcription start site by using 5' and 3' deletions of the P5 promoter fused to the chloramphenicol acetyltransferase gene. This element contains four imperfect GAGC repeats similar to the Rep recognition sequences (RRSs) in the AAV inverted terminal repeats and in the AAV preferred integration locus in chromosome 19. Band shift analyses showed that human 293 cell nuclear extracts containing Rep68 or Rep68/K340H, a putative nucleoside triphosphate (NTP)-binding-site mutant of Rep68, formed Rep-specific complexes with this P5 RRS DNA. Within the P5 RRS, mutation of a cytosine at position 273 in the AAV sequence to guanine abolished Rep68 binding to the DNA. A mutation in the P5 RRS within a full-length AAV genome, which abolished Rep binding, resulted in a 40 to 50% reduction in the ability of wild-type Rep68 to inhibit the accumulation of P5 transcripts in vivo. In contrast, the Rep68/K340H mutant was unable to down-regulate this mutated promoter. These results indicate that there are at least two mechanisms involved in the negative regulation of P5 transcript levels by Rep68; one involves Rep68 binding to the P5 RRS, and another requires the region of Rep68 containing the consensus NTP-binding motif. Furthermore, our studies of AAV genomes containing mutated RRS- and/or YY1-binding elements suggest that transcription factor YY1 binding to the transcription start site of P5 interferes with Rep68 repression of the P5 promoter.
The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP-Rep68 delta) has previously been shown to possess DNA-DNA helicase activity, as does the purified wild-type Rep68. In the present study, we demonstrate that MBP-Rep68 delta also catalyzes the unwinding of a DNA-RNA hybrid. MBP-Rep68 delta-mediated DNA-RNA helicase activity required ATP hydrolysis and the presence of Mg2+ ions and was inhibited by high ionic strength. The efficiency of the DNA-RNA helicase activity of MBP-Rep68 delta was comparable to its DNA-DNA helicase activity. However, MBP-Rep68 delta lacked the ability to unwind a blunt-ended DNA-RNA substrate and RNA-RNA duplexes. We have also demonstrated that MBP-Rep68 delta has ATPase activity which is enhanced by the presence of single-stranded DNA but not by RNA. The MBP-Rep68 delta NTP mutant protein, which has a lysine-to-histidine substitution at amino acid 340 in the putative nucleoside triphosphate-binding site of Rep68, not only lacks DNA-RNA helicase and ATPase activities but also inhibits the helicase activity of MBP-Rep68 delta. DNA-RNA helicase activity of Rep proteins might play a pivotal role in the regulation of AAV gene expression by AAV Rep proteins.
AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration.
Replication of the palindromic inverted terminal repeats (ITRs) of adeno-associated virus type 2 requires several functions of the viral nonstructural Rep proteins. These include binding to the ITR, nicking of the double-stranded replication intermediate at the terminal resolution site (trs), and then strand displacement and synthesis from the nick. This report demonstrates the ability of both recombinant fusion maltose-binding protein (MBP)-Rep68 delta produced in Escherichia coli and wild-type (wt) Rep68 to bind to a linear truncated form of the ITR, delta 57 ITR, with similar affinity as to the wt hairpin ITR. A dissociation constant for MBP-Rep68 delta of approximately 8 x 10(-10) M was determined for the wt ITR and delta 57 ITR probes. Truncation of delta 57 ITR to generate delta 28 ITR, which retains the GCTC repeat motif but not the trs, bound at least 10 times less efficiently than delta 57 ITR. Extension of delta 28 ITR with nonspecific sequence restored the ability of MBP-Rep68 delta to bind to delta 28 ITR. Thus, high-affinity binding would appear to require stabilization by flanking sequence as well as the intact GCTC repeat motif. Cleavage of the delta 57 ITR probe with DdeI, which truncates the flanking sequence and was previously shown to inhibit binding by Rep68, also inhibited the binding of MBP-Rep68 delta. The requirements for stable binding were further defined with a series of oligonucleotide probes which spanned the region protected by MBP-Rep78 in DNase I footprinting. The binding activity of either MBP-Rep68 delta or wt Rep68 to hairpin ITR or delta 57 ITR was indistinguishable. However, the binding activity of MBP-Rep68 delta to DNA does not appear to correlate with trs endonuclease activity. The nicking and covalent linkage of MBP-Rep68 delta to the nonhairpin delta 57 ITR was approximately 100-fold less efficient than its linkage to a hairpin-containing ITR. Therefore, although the hairpin portion of the ITR does not appear to play a role in recognition and stabilization of MBP-Rep68 delta binding, its presence does affect the trs cleavage activity of the protein.
Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.
The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence of the p5 promoter.