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W A Miller

Publications and source records attributed to W A Miller.

At least 37 records · Page 2Linked to original sources
PubMed1999

Primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic RNA1.

Barley yellow dwarf luteovirus (BYDV) generates three 3'-coterminal subgenomic RNAs (sgRNAs) in infected cells. The promoter of sgRNA1 is a putative hot spot for RNA recombination in luteovirus evolution. The sgRNA1 transcription start site was mapped previously to either nucleotide 2670 or nucleotide 2769 of BYDV genomic RNA (gRNA) in two independent studies. Our data support the former initiation site. The boundaries of the sgRNA1 promoter map between nucleotides 2595 and 2692 on genomic RNA. Computer prediction, phylogenetic comparison, and structural probing revealed two stem-loops (SL1 and SL2) in the sgRNA1 promoter region on the negative strand. Promoter function was analyzed by inoculating protoplasts with a full-length infectious clone of the BYDV genome containing mutations in the sgRNA promoter. Because the promoter is located in an essential coding region of the replicase gene, we duplicated it in a nonessential part of the genome from which a new sgRNA was expressed. Mutational analysis revealed that secondary structure, but not the nucleotide sequence, was important at the base of SL1. Regions with both RNA primary and secondary structural features that contributed to transcription initiation were found at the top of SL1. Primary sequence, but not the secondary structure, was required in SL2, which includes the initiation site. Disruption of base pairing near the sgRNA1 start site increased the level of transcription three- to fourfold. We propose that both primary and secondary structures of the sgRNA1 promoter of BYDV play unique roles in sgRNA1 promoter recognition and transcription initiation.

Base Sequence↗
PubMed1998

Screening of maternal serum for fetal Down's syndrome in the first trimester.

BACKGROUND: Screening of maternal serum to identify fetuses with Down's syndrome is now routinely offered during the second trimester of pregnancy. Prenatal screening by means of serum assays or ultrasonographic measurements, either alone or in combination, may also be possible in the first trimester. METHODS: We measured serum alpha-fetoprotein, unconjugated estriol, human chorionic gonadotropin (hCG), the free beta subunit of hCG, and pregnancy-associated protein A in 4412 women (82 percent of whom were 35 years of age or older) who came to 16 prenatal diagnostic centers for chorionic-villus sampling or early amniocentesis at 9 to 15 weeks of gestation. Ultrasound measurements of fetal nuchal translucency were also reported. Fetal chromosomal analysis was performed in all pregnancies. Altogether, there were 61 fetuses with Down's syndrome. RESULTS: A total of 48 pregnancies affected by Down's syndrome and 3169 unaffected pregnancies were identified before 14 weeks of gestation; the rates of detection of Down's syndrome for the five serum markers were as follows: 17 percent for alpha-fetoprotein, 4 percent for unconjugated estriol, 29 percent for hCG, 25 percent for the free beta subunit of hCG, and 42 percent for pregnancy-associated protein A, at false positive rates of 5 percent. The results of the measurements of serum hCG and its free beta subunit were highly correlated. When used in combination with the serum concentration of pregnancy-associated protein A and maternal age, the detection rate was 63 percent for hCG (95 percent confidence interval, 47 to 76 percent) and 60 percent for its free beta subunit (95 percent confidence interval, 45 to 74 percent). Measurements of nuchal translucency varied considerably between centers and could not be reliably incorporated into our calculations. CONCLUSIONS: Screening for Down's syndrome in the first trimester is feasible, with use of measurements of pregnancy-associated protein A and either hCG or its free beta subunit in maternal serum.

Adolescent↗
PubMed1997

A viral sequence in the 3'-untranslated region mimics a 5' cap in facilitating translation of uncapped mRNA.

For recognition by the translational machinery, most eukaryotic cellular mRNAs have a 5' cap structure [e.g. m7G(5')ppp(5')N]. We describe a translation enhancer sequence (3'TE) located in the 3'-untranslated region (UTR) of the genome of the PAV barley yellow dwarf virus (BYDV-PAV) which stimulates translation from uncapped mRNA by 30- to 100-fold in vitro and in vivo to a level equal to that of efficient capped mRNAs. A four base duplication within the 3'TE destroyed the stimulatory activity. Efficient translation was recovered by addition of a 5' cap to this mRNA. Translation of both uncapped mRNA containing the 3'TE in cis and capped mRNA lacking any BYDV-PAV sequence was inhibited specifically by added 3'TE RNA in trans. This inhibition was reversed by adding initiation factor 4F (eIF4F), suggesting that the 3'TE, like the 5' cap, mediates eIF4F-dependent translation initiation. The BYDV-PAV 5'UTR was necessary for the 3'TE to function, except when the 3'TE itself was moved to the 5'UTR. Thus, the 3'TE is sufficient for recruiting the translation factors and ribosomes, while the viral 5'UTR may serve only for the long distance 3'-5' communication. Models are proposed to explain this novel mechanism of cap-independent translation initiation facilitated by the 3'UTR.

Conserved Sequence↗
PubMed1997

The satellite RNA of barley yellow dwarf virus-RPV is supported by beet western yellows virus in dicotyledonous protoplasts and plants.

The subgroup II luteovirus barley yellow dwarf virus-RPV (BYDV-RPV) acts as a helper virus for a satellite RNA (satRPV RNA). The subgroup II luteovirus beet western yellows virus (BWYV) and the ST9-associated RNA (ST9a RNA), a BWYV-associated RNA that encodes a polymerase similar to those of subgroup I luteoviruses, were assayed for their ability to support replication of satRPV RNA. SatRPV RNA was replicated in tobacco protoplasts in the presence of BWYV RNA or a mixture of BWYV plus the ST9a RNA, but not in the presence of ST9a RNA alone. ST9a RNA stimulated BWYV RNA accumulation which, in turn, increased the accumulation of satRPV RNA. SatRPV RNA was encapsidated in BWYV capsids primarily as circular monomers, which differs from the linear monomers found in BYDV (RPV + PAV) particles. SatRPV RNA was transmitted to Capsella bursa-pastoris plants by aphids only in the presence of BWYV and ST9a RNA. SatRPV RNA reduced accumulation of both BWYV helper and ST9a nonhelper RNAs in plants but did not affect symptoms. The replication of satRPV RNA only in the presence of subgroup II luteoviral RNAs but not in the presence of RNAs with subgroup I-like polymerase genes, in both monocotyledonous and dicotyledonous hosts, suggests that the specificity determinants of satRPV RNA replication are contained within the polymerase genes of supporting viruses rather than in structural genes or host plants.

Animals↗
PubMed1997

Barley yellow dwarf viruses.

Barley yellow dwarf viruses represent one of the most economically important and ubiquitous groups of plant viruses. This review focuses primarily on four research areas in which progress has been most rapid. These include (a) evidence supporting reclassification of BYDVs into two genera; (b) elucidation of gene function and novel mechanisms controlling gene expression; (c) initial forays into understanding the complex interactions between BYDV virions and their aphid vectors; and (d) replication of a BYDV satellite RNA. Economic losses, symptomatology, and means of control of BYD are also discussed.

Journal Article↗
PubMed1996

Aphid transmission and systemic plant infection determinants of barley yellow dwarf luteovirus-PAV are contained in the coat protein readthrough domain and 17-kDa protein, respectively.

Proteins encoded by open reading frames (ORF) 3, 4, and 5 of the barley yellow dwarf luteovirus genome are translated from a single subgenomic RNA. The structural proteins are encoded by ORF 3 (coat protein) and ORF 5 (readthrough domain) and contain undefined domains that regulate the movement of virus through aphid vectors. The biological function of the nonstructural 17-kDa protein encoded by ORF 4 is unknown. A complementation method was employed to test the ability of barley yellow dwarf virions carrying mutations within the readthrough domain and the 17-kDa protein to be transmitted by aphids and to cause systemic infections in plants. We show that the readthrough domain is required for aphid transmission; however, it is not required for virus to be taken up by aphid hindgut cells and released into the hemocoel. The circulative pathway of luteoviruses in aphid vectors requires that virus be actively transported from the hemolymph into the salivary system. Thus, it appears that the readthrough domain is required for transport of virus through membranes of the aphid salivary glands. Furthermore, the readthrough domain was not required for systemic infection of plants, but did influence the accumulation of virus in infected plants. The 17-kDa protein is required for the systemic infection of plants.

Animals↗
PubMed1996

Incidence and significance of chromosome mosaicism involving an autosomal structural abnormality diagnosed prenatally through amniocentesis: a collaborative study.

Among 179,663 prenatal diagnosis cases collected from ten institutions and two publications, 555 (0.3 per cent) were diagnosed as having chromosome mosaicism. Of these, 57 (10.3 per cent) were mosaic for an autosomal structural abnormality, 28 (5 per cent) for a sex chromosome structural abnormality, and 85 (15.3 per cent) were mosaic for a marker chromosome. Ninety-five cases of prenatally diagnosed mosaicism with a structural abnormality in an autosome and a normal cell line, and with a known phenotypic outcome, were collected for karyotype-phenotype correlations through our collaboration (40 cases), a prior survey (26 cases), and published reports (29 cases). They included 13 balanced reciprocal translocations, one unbalanced reciprocal translocation, four balanced Robertsonian translocations, four unbalanced Robertsonian translocations, four inversions, 17 deletions, three ring chromosomes, 19 i(20q), seven +i(12p), six other isochromosomes, and 17 partial trisomies resulting from a duplication or other rearrangement. All cases mosaic for a balanced structural rearrangement resulted in a normal phenotype. All cases of 46/46,i(20q) resulted in normal liveborns. Five of seven cases with 46/47,+i(12p) had an abnormal phenotype compatible with Killian-Pallister syndrome. The overall risk for an abnormal outcome for a mosaic case with an unbalanced structural abnormality, excluding 46/46,i(20q) and 46/47,+i(12p), is 40.4 per cent. In the same category, the study also suggested a correlation between the percentage of abnormal cells and an abnormal phenotype. For mosaicism involving a terminal deletion, the possibility of a familial fragile site should be considered.

Amniocentesis↗
PubMed1996

Local and distant sequences are required for efficient readthrough of the barley yellow dwarf virus PAV coat protein gene stop codon.

Many viruses use stop codon readthrough as a strategy to produce extended coat or replicase proteins. The stop codon of the barley yellow dwarf virus (PAV serotype) coat protein gene is read through at a low rate. This produces an extended polypeptide which becomes part of the virion. We have analyzed the cis-acting sequences in the barley yellow dwarf virus PAV genome required for this programmed readthrough in vitro in wheat germ extracts and reticulocyte lysates and in vivo in oat protoplasts. Two regions 3' to the stop codon were required. Deletion of sections containing the first 5 of the 16 CCN NNN repeats located 3' of the stop codon greatly reduced readthrough in vitro and in vivo. Surprisingly, readthrough also required a second, more distal element that is located 697 to 758 bases 3' of the stop codon within the readthrough open reading frame. This element also functioned in vivo in oat protoplasts when placed more than 2 kb from the coat protein gene stop in the untranslated region following a GUS reporter gene. This is the first report of a long-range readthrough signal in viruses.

Avena↗
PubMed1995

Genes and cis-acting sequences involved in replication of barley yellow dwarf virus-PAV RNA.

Deletion and point mutation analyses were employed to determine gene products and cis-acting signals involved in translation, replication, and encapsidation of barley yellow dwarf virus (PAV serotype) RNA in oat protoplasts. Of the six open reading frames (ORFs), only ORFs 1 and 2, which include the putative RNA-dependent RNA polymerase gene, were required for replication. In vitro translation of these mutants revealed that sequence upstream of the shifty heptanucleotide was required for ribosomal frameshifting, and that a 3'-translational enhancer stimulated translation more efficiently when located in closer proximity to the translated ORFs. Deletion of the coat protein gene reduced the accumulation of genomic but not subgenomic RNA. The carboxy-terminally extended form of the coat protein, produced by readthrough of its stop codon, was not required for encapsidation. Although the ORF6 product was not necessary, cis-acting RNA signals in and around ORF6 were required for RNA replication. Defective RNAs harboring various deletions were not replicated in trans by the co-inoculated wild-type helper genome, suggesting that replication of PAV RNA may be coupled to translation.

Avena↗
PubMed1995

A sequence located 4.5 to 5 kilobases from the 5' end of the barley yellow dwarf virus (PAV) genome strongly stimulates translation of uncapped mRNA.

An infectious, in vitro transcript from a full-length cDNA clone of the barley yellow dwarf virus (PAV serotype) genome translated efficiently in a wheat germ translation extract. Deletions in a region that we call the 3' translational enhancer, located between bases 4,513 and 5,009 in the 5,677-base genome, reduced translation of the 5'-proximal open reading frames from uncapped RNA by at least 30-fold. Deletions elsewhere in all but the 5' end of the genome had no effect on translation. Presence of a m7G(5')pp(5')G cap on the 5' end fully restored translational efficiency of transcripts lacking the 3' translational enhancer. The translation enhancer reduced inhibition of translation by free cap analog, did not affect RNA stability, and did not function in reticulocyte lysates. When placed in the 3'-untranslated region of uncapped mRNA encoding the beta-glucuronidase gene, the translation enhancer stimulated translation more than 80-fold, in the presence of the viral, but not a plasmid-derived, 5' leader. A polyadenylate tail could not substitute for the 3' translation enhancer. These observations provide an extreme example, in terms of distance from the 5' end and level of stimulation, of an mRNA in which a sequence near the 3' end stimulates translation.

Enhancer Elements, Genetic↗
PubMed1995

Does gender have an impact on the sonographic detection of second-trimester fetuses with Down's syndrome?

The biometric and structural sonographic features of 95 second-trimester fetuses with Down's syndrome were evaluated to determine whether affected male fetuses differed from affected females. There were 54 male and 41 female fetuses with Down's syndrome studied. A shortened femur was identified in 28/54 (52%) males compared with 19/41 (46%) affected females (NS). A thickened nuchal fold was identified in 19/54 (35%) of males vs. 20/41 (49%) of females. Renal pyelectasis was seen in 7/54 (13%) males and 8/41 (19%) females. A heart defect was seen in 8/54 (15%) males and 7/41 (17%) females. Ventriculomegaly was identified in 6/54 (11%) males and 3/41 (7%) females with Down's syndrome. There were no statistically significant differences in the incidence of the sonographic findings when male and female Down's fetuses were compared. Our data show that the criteria for evaluation of sonographic markers for the identification of second-trimester fetuses with Down's syndrome should be the same in male and female fetuses.

Abnormalities, Multiple↗
PubMed1995

Fetal liver calcifications: sonographic appearance and postnatal outcome.

PURPOSE: To determine the importance of intrahepatic calcifications seen sonographically in the fetus. MATERIALS AND METHODS: The authors retrospectively reviewed sonograms obtained in fetuses scanned between 16 and 40 gestational weeks in whom intrahepatic calcifications were seen. The number and character of these calcifications were determined. Follow-up scans obtained in utero and after birth were also reviewed. Outcome was determined after birth. RESULTS: Thirty-three fetuses had intrahepatic calcifications at 16-38 weeks gestation. Four of the 33 fetuses died, one of which had liver calcifications as the only sonographic finding. This fetus had cytomegalovirus and was the only fetus with increasing numbers of calcifications on follow-up scans. All fetuses with isolated liver calcifications had a normal postnatal outcome (96% survival rate for fetuses with isolated intrahepatic calcifications). Of eight fetuses with additional anomalies, five lived; only two were healthy newborns. CONCLUSION: The outcome in fetuses with isolated intrahepatic calcifications is usually excellent, although viral causes must be excluded if additional findings appear.

Abnormalities, Multiple↗
PubMed1994

Replication of barley yellow dwarf virus satellite RNA transcripts in oat protoplasts.

A small RNA associated with an isolate of barley yellow dwarf virus (BYDV) has been described which has the physical properties of a satellite RNA (Miller et al., Virology 183, 711-720, 1991). Here we demonstrate that this RNA has the biological properties of a satellite RNA: it requires the presence of helper virus (BYDV genomic) RNA for replication and the helper RNA does not require the satellite. To obtain pure satellite RNA, a permuted dimeric clone was constructed from which infectious satellite RNA could be transcribed in vitro. The dimeric transcript self-cleaved to produce monomeric satellite RNA. When this RNA was coelectroporated with BYDV genomic RNA into oat protoplasts, replication of both RNAs was detected by Northern hybridization. The RPV, but not the PAV, serotype of BYDV supported satellite RNA replication. The presence of discrete oligomeric forms of (+) and (-) sense satellite RNA in infected protoplasts suggests that both strands replicate by a rolling circle mechanism.

Base Sequence↗