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M Janda

Publications and source records attributed to M Janda.

30 records · Page 2Linked to original sources
PubMed1993

RNA-dependent replication, transcription, and persistence of brome mosaic virus RNA replicons in S. cerevisiae.

This paper shows that yeast can be a host for the replication of a higher eukaryotic viral genome. Brome mosaic virus (BMV) is a member of the alphavirus-like superfamily of animal and plant positive strand RNA viruses. Yeast expressing BMV RNA replication genes 1a and 2a support RNA-dependent replication and transcription of BMV RNA3 derivatives. RNA3 derivatives with the coat gene replaced by a reporter gene expressed that gene in a fashion dependent on 1a and 2a expression in trans and on BMV RNA replication and transcription signals in cis. RNA3 derivatives carrying the yeast URA3 gene complemented ura3- yeast to prototrophy and were maintained as persistent RNA episomes. Thus, all cellular factors essential for BMV RNA replication and transcription must be present in yeast, and yeast genetics should facilitate their identification.

Base Sequence↗
PubMed1993

Bromovirus RNA replication and transcription require compatibility between the polymerase- and helicase-like viral RNA synthesis proteins.

The positive-strand RNA bromoviruses encode two nonstructural proteins, 1a and 2a, involved in RNA-dependent RNA replication. These proteins have extensive sequence similarities with methyltransferase, helicase, and polymerase proteins of other plant and animal viruses. 1a and 2a can also form a complex in vitro. To explore whether 1a-2a interaction is required for RNA replication in vivo, we reassorted the 1a and 2a genes from two different bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV). 1a and 2a were expressed independently of viral replication by using RNA- or DNA-based transient expression, and their in vivo RNA replication activities were tested in protoplasts with BMV and CCMV RNA3 templates. RNA-based transient expression confirmed prior indications that bromovirus RNA replication is more sensitive to reductions in 1a expression than to reductions in 2a expression. DNA-based expression of the homologous combinations of 1a and 2a supported high levels of RNA synthesis, but both 1a-2a heterologous combinations exhibited RNA synthesis defects. The combination of CCMV 1a and BMV 2a did not support detectable synthesis of negative-strand, positive-strand, or subgenomic RNA. The converse combination of BMV 1a and CCMV 2a was preferentially defective in positive-strand and subgenomic RNA accumulation, showing that 1a-2a interaction is involved in these processes in ways distinct from negative-strand RNA synthesis, which was only slightly affected. These results indicate that at least some functions of 1a and 2a operate in a mutually dependent manner in vivo and that the mechanisms of positive- and negative-strand RNA synthesis are differentiated in part by features of such interactions.

Base Sequence↗
PubMed1993

Bromovirus movement protein genes play a crucial role in host specificity.

Monocot-adapted brome mosaic virus (BMV) and dicot-adapted cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes. Although RNAs 1 and 2 together are sufficient for RNA replication in protoplasts, systemic infection also requires RNA3, which encodes the coat protein and the nonstructural 3a movement protein. We have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by RNA3 as well as by RNA1 and/or RNA2. Here, to test their possible role in host specificity, the 3a movement protein genes were precisely exchanged between BMV and CCMV. The hybrid viruses, but not 3a deletion mutants, systemically infected Nicotiana benthamiana, a permissive host for both parental viruses. The hybrids thus retain basic competence for replication, packaging, cell-to-cell spread, and long-distance (vascular) spread. However, the hybrids failed to systemically infect either barley or cowpea, selective hosts for parental viruses. Thus, the 3a gene and/or its encoded 3a protein contributes to host specificity of both monocot- and dicot-adapted bromoviruses. Tests of inoculated cowpea leaves showed that the spread of the CCMV hybrid containing the BMV 3a gene was blocked at a very early stage of infection. Moreover, the BMV hybrid containing the CCMV 3a gene appeared to spread farther than wt BMV in inoculated cowpea leaves. Several pseudorevertants directing systemic infection in cowpea leaves were obtained from plants inoculated with the CCMV(BMV 3a) hybrid, suggesting that the number of mutations required to adapt the hybrid to dicots is small.

Base Sequence↗
PubMed1989

Sequence of cowpea chlorotic mottle virus RNAs 2 and 3 and evidence of a recombination event during bromovirus evolution.

The genomic sequence of cowpea chlorotic mottle virus (CCMV) was completed by sequencing biologically active cDNA clones of CCMV RNA2 (2774 bases) and RNA3 (2173 bases). While only the central core of the encoded 94-kDa CCMV 2a protein contains features conserved among known and putative RNA replication proteins from many viruses, both flanking regions of CCMV 2a show substantial similarity to the corresponding protein of the related brome mosaic virus (BMV). The 3a proteins of CCMV and BMV, implicated as contributors to the distinct host specificities of the two viruses, show lower levels of conservation but are still discernibly related throughout. Major differences occur in the organization of noncoding sequences in CCMV and BMV RNA3. With respect to an otherwise similar region preceding the BMV 3a gene, the CCMV RNA3 5' noncoding sequence contains a clearly bounded 111-base insertion that must reflect a sequence rearrangement in evolution of at least one of the two viruses. The presence of a subgenomic promoter-like sequence near the end of the novel CCMV sequence makes the organization of genes in CCMV RNA3 reminiscent of the 3' end of tobacco mosaic virus RNA, suggesting that CCMV or its 3a gene might have been derived from an ancestor with fewer genomic RNAs. Sequence similarities between the CCMV and BMV RNA3 intercistronic regions include the subgenomic mRNA promoter and an oligo(A), but not an intercistronic segment required for BMV RNA3 amplification, implying that replication signals on the two RNA3s may be organized quite differently.

Amino Acid Sequence↗
PubMed1988

Infectious in vitro transcripts from cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA components with brome mosaic virus.

Complete cDNA copies of genomic RNA1, RNA2, and RNA3 of cowpea chlorotic mottle virus (CCMV) were cloned 1 base downstream from a T7 RNA polymerase promoter. The mixture of capped in vitro transcripts from all three clones produced normal CCMV infections in barley protoplasts and cowpea plants. By using transcripts from these clones and from a similar set of biologically active clones of the related brome mosaic virus (BMV), all possible single component exchanges between the BMV and CCMV tripartite genomes were tested. Viral RNA replication was not observed with any heterologous combination of RNA1 and RNA2, which encode trans-acting viral RNA replication factors. However, substitution of the heterologous RNA3 into either genome produced viable hybrid viruses, both of which replicated in barley protoplasts and produced lesions on Chenopodium hybridum, a local lesion host for both parent viruses. In hybrid infections, BMV and CCMV coat proteins each readily packaged RNAs from the heterologous virus, but BMV RNAs were replicated to a higher level than CCMV RNAs, even when trans-acting RNA replication factors were provided by CCMV genes. Neither hybrid systemically infected the natural host of either parent virus, suggesting that host specificity determinants in BMV and CCMV are encoded by RNA3 and at least one other genomic RNA.

Base Sequence↗
PubMed1984

Multicomponent RNA plant virus infection derived from cloned viral cDNA.

In vitro transcripts from mixtures of appropriate brome mosaic virus (BMV) cDNA clones are infectious when inoculated onto barley plants. Infectivity depends on in vitro transcription and on the presence of transcripts from clones of all three BMV genetic components. Infectivity is destroyed by RNase after transcription, but it is insensitive to RNase before or to DNase after transcription. Virion RNAs from plants infected with cDNA transcripts hybridize to BMV-specific probes and coelectrophorese with virion RNAs propagated from conventional inoculum. Direct RNA sequencing shows that a deletion in the noncoding region of one infectious BMV clone is preserved in viral RNA from plants systemically infected with transcript mixtures representing that clone.

Journal Article↗
PubMed1984

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome.

Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site. After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs. Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence. When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs. Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs. pPM1 should facilitate in vitro production of other viral and nonviral RNAs.

Base Sequence↗
PubMedDate not supplied

Primary fallopian tube carcinoma: the Queensland experience.

The purpose of this study was to review the experience with fallopian tube carcinoma in Queensland and to compare it with previously published data. Thirty-six patients with primary fallopian tube carcinoma treated at the Queensland Gynaecological Cancer Center from 1988 to 1999 were reviewed in a retrospective clinicopathologic study. All patients had primary surgery and 31/36 received chemotherapy postoperatively. Abnormal vaginal bleeding (15/36) and abdominal pain (14/36) were the most common presenting symptoms at the time of diagnosis. Median follow-up was 70.3 months and the median overall survival was 68.1 months. Surgical stage I disease (P = 0.02) and the absence of residual tumor after operation (P = 0.03) were the only factors associated with improved survival. Twenty of the 36 patients (55%) presented with stage I disease and survival was 62.7% at 5 years. No patient with postoperative residual tumor survived. The majority of the patients with fallopian tube carcinoma present with stage I disease at diagnosis, but their survival probability is low compared with that of other early stage gynecological malignancies. If primary surgical debulking cannot achieve macroscopic tumor clearence, the chance of survival is extremely low.

Abdominal Pain↗
PubMedDate not supplied

Anemia before and during concurrent chemoradiotherapy in patients with cervical carcinoma: Effect on progression-free survival.

To determine the impact of anemia before and during chemoradiation in patients with cervical cancer, we collected data on hemoglobin (Hb) levels before and during treatment from 60 unselected patients with cervical carcinoma. All patients had FIGO stage IB to IVA disease and were treated with concurrent chemoradiation for the aim of cure. Patients with an Hb value below or equal to the lower 25th quartile were considered anemic. Progression-free survival (PFS) was evaluated by univariate and multivariate analyses. After a median follow-up of 26.3 months, 20 patients developed disease progression. The lowest Hb during chemoradiation (nadir Hb), the stage of disease, and parametrial involvement were correlated significantly with PFS. On multivariate analysis, the nadir Hb (relative risk [RR] 0.29) and tumor stage (RR 3.4) remained the only prognostically relevant factors predicting PFS. At 60 months the PFS was 39.1% for anemic patients and 48.0% for nonanemic patients (P < 0.0002). In patients undergoing chemoradiation for cervical carcinoma, a low nadir Hb is highly predictive of shortened PFS, whereas the Hb before treatment is prognostically not significant.

Adenocarcinoma↗
PubMedDate not supplied

Vulvar cancer patients' quality of life: a qualitative assessment.

To develop a vulvar cancer-specific quality of life (QOL) subscale to accompany the Functional Assessment of Cancer-General (FACT-G) questionnaire, semistructured interviews were performed with 15 patients treated for vulvar cancer (FIGO stage 0-3). All but one patient, who received chemoradiotherapy, were treated by radical vulvectomy and six patients received a groin lymph node dissection. Patients experienced reductions in several aspects of QOL including emotional functioning, physical functioning, social functioning, sexuality, and body image. Six patients suffered from lymphedema of the legs with a mean severity of 3.5 on a 10-point scale. Four patients reported pruritus (severity rating 8.5). Seven patients expressed a need for more information about the illness and treatment. Only four patients returned to employment after treatment, and all of these patients reported work-related problems. Reductions in sexual functioning were a major concern for five patients, all younger than 65 years. Other topics were groin discomfort after removal of the lymph nodes and disturbance by odor from the vulva. Results of this study revealed vulvar cancer-specific reductions in QOL for inclusion in the newly developed vulvar cancer-specific subscale.

Age Factors↗