30-kDa Gene Research Articles

Molecular prevalence and associated risk factors of Theileria annulata infections in dromedary camels in Egypt.

Theileria annulata (T. annulata) is an intracellular protozoan, transmitted by ixodid ticks of the genus Hyalomma and affects camels. There are few epidemiological data on T. annulata infection and its associated risk factors in Egyptian camels. Thus, the present study aimed to determine the prevalence of T. annulata in camels using PCR and assess the associated risk factors for infection. A total of 380 blood samples were collected from camels raising in three Egyptian governorates and examined by PCR assay targeting 30-kDa gene to detect the presence of T. annulata infection, beside statistical analysis of associated factors. The results revealed presence of T. annulata with overall prevalence of 21.1%. In addition, the univariate analysis revealed significant (P<0.05) association between prevalence of T. annulata in camels and locality, age, sex, tick infestation, and application of acaricides. Whereas the prevalence of T. annulata was higher in camels of age group >6 to 10 years (38%), females (25.7%) and in infested camels with ticks (29%) and in case of absence of acaricides application (25%). In contrast, the body condition of camels had not significant effect on prevalence of theileriosis in camels. The current study concluded that T. annulata is prevalent in Egyptian camels and that a tick control program is required to reduce the risk of infection.

The Saccharomyces cerevisiae HCR1 Gene Encoding a Homologue of the p35 Subunit of Human Translation Initiation Factor 3 (eIF3) Is a High Copy Suppressor of a Temperature-sensitive Mutation in the Rpg1p Subunit of Yeast eIF3

The complex eukaryotic initiation factor 3 (eIF3) was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary complex, and aiding mRNA binding to 40 S ribosomal subunits. Recently, we described the identification of RPG1 (TIF32), the p110 subunit of the eIF3 core complex in yeast. In a screen for Saccharomyces cerevisiae multicopy suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified. When overexpressed, the 30-kDa gene product, named Hcr1p, was able to support, under restrictive conditions, growth of the rpg1-1 temperature-sensitive mutant, but not of a Rpg1p-depleted mutant. An hcr1 null mutant was viable, but showed slight reduction of growth when compared with the wild-type strain. Physical interaction between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation analysis. The combination of Deltahcr1 and rpg1-1 mutations resulted in a synthetic enhancement of the slow growth phenotype at a semipermissive temperature. In a computer search, a significant homology to the human p35 subunit of the eIF3 complex was found. We assume that the yeast Hcr1 protein participates in translation initiation likely as a protein associated with the eIF3 complex.

Effects of Coat Protein Mutations and Reduced Movement Protein Expression on Infection Spread by Cowpea Chlorotic Mottle Virus and Its Hybrid Derivatives

Previously we have reported that the essential 3a movement gene of icosahedral cowpea chlorotic mottle virus (CCMV) can be functionally replaced by the 30-kDa movement gene of rod-shaped sunn-hemp mosaic virus (SHMV). Because plant RNA viruses differ in requiring or not requiring coat protein for systemic infection, we have now investigated whether systemic spread by this CCMV/SHMV hybrid is dependent on its CCMV coat protein as well as its SHMV movement protein. We find that either deletion or frameshift mutations in the coat protein gene block systemic spread. Thus, like wild-type CCMV, systemic infection by the hybrid is dependent on both movement protein and coat protein. These results further support the conclusion that the required functions of the coat and movement proteins in CCMV spread do not depend on sequence-specific interaction between these proteins. Additional features of the hybrid also motivated testing the effects of modulating movement protein expression. Creating an extra, out-of-frame translational start codon (AUG) shortly upstream of the 3a movement protein gene in CCMV downregulated its expression 18-fold. Nevertheless, for CCMV derivatives bearing either the CCMV 3a gene or the SHMV 30-kDa gene, the extra AUG resulted in only a minor delay in the onset of viral spread and little or no effect on the subsequent rate of cell-to-cell spread. Thus, under normal circumstances, the rate of CCMV cell-to-cell spread in cowpea plants appears to be limited primarily by factors other than movement protein synthesis.

Viral RNA trafficking is inhibited in replicase-mediated resistant transgenic tobacco plants.

Transgenic tobacco (Nicotiana tabacum cv. Turkish Samsun NN) plants expressing a truncated replicase gene sequence from RNA-2 of strain Fny of cucumber mosaic virus (CMV) are resistant to systemic CMV disease. This is due to suppression of virus replication and cell-to-cell movement in the inoculated leaves of these plants. In this study, microinjection protocols were used to directly examine cell-to-cell trafficking of CMV viral RNA in these resistant plants. CMV RNA fluorescently labeled with the nucleotide-specific TOTO-1 iodide dye, when coinjected with unlabeled CMV 3a movement protein (MP), moved rapidly into the surrounding mesophyll cells in mature tobacco leaves of vector control and untransformed plants. Such trafficking required the presence of functional CMV 3a MP. In contrast, coinjection of CMV 3a MP and CMV TOTO-RNA failed to move in transgenic resistant plants expressing the CMV truncated replicase gene. Furthermore, coinjection of 9.4-kDa fluorescein-conjugated dextran (F-dextran) along with unlabeled CMV 3a MP resulted in cell-to-cell movement of the F-dextran in control plants, but not in the transgenic plants. Similar results were obtained with viral RNA when the 30-kDa MP of tobacco mosaic virus (TMV) was coinjected with TMV TOTO-RNA into replicase-resistant transgenic tobacco expressing the 54-kDa gene sequence of TMV. However, in these transgenic plants, the TMV-MP was still capable of mediating cell-to-cell movement of itself and the 9.4-kDa F-dextran. These results indicate that an inhibition of cell-to-cell viral RNA trafficking is correlated with replicase-mediated resistance. This raises the possibility that the RNA-2 product is potentially involved in the regulation of cell-to-cell movement of viral infectious material during CMV replication.

Resistance to Pepper Mild Mottle Tobamovirus Conferred by the 54-kDa Gene Sequence in Transgenic Plants Does Not Require Expression of the Wild-Type 54-kDa Protein

We previously reported thatNicotiana benthamianaplants transformed with the wild-type 54-kDa region of the pepper mild mottle tobamovirus, S strain (PMMoV-S), displayed two different resistance responses against PMMoV infection. Some of the transgenic plants exhibited a complete and highly resistant phenotype while the remaining plants showed a delayed resistance (Tenlladoet al.,1995,Virology211, 170–183). Here we show that some of theN. benthamianaplants transformed with a construct expressing a PMMoV-S truncated 54-kDa protein coding sequence also displayed a complete and highly resistant phenotype similar to that shown by the wild-type 54-kDa transgenic plants. This result indicates that the wild-type, full-length 54-kDa protein is not required in mediating the complete resistance phenotype against PMMoV. The remaining truncated 54-kDa transgenic plants were susceptible to PMMoV infection but showed a variable delay in the appearance of symptoms. Unlike the wild-type 54-kDa transgenic plants, which were initially susceptible to the infection but recovered later, the truncated 54-kDa transgenic plants never exhibited this delayed resistance phenotype. However, they displayed a new type of altered symptomatic phenotype. The truncated 54-kDa transgenic lines also exhibited a lower level of transgenic transcripts compared to the wild-type 54-kDa transgenic lines which could account for the absence of the delayed resistance phenotype.

Sequencing and analysis of the prolate-headed lactococcal bacteriophage c2 genome and identification of the structural genes.

The 22,163-bp genome of the lactococcal prolate-headed phage c2 was sequenced. Thirty-nine open reading frames (ORFs), early and late promoters, and a putative transcription terminator were identified. Twenty-two ORFs were in the early gene region, and 17 were in the late gene region. Putative genes for a DNA polymerase, a recombination protein, a sigma factor protein, a transcription regulatory protein, holin proteins, and a terminase were identified. Transcription of the early and late genes proceeded divergently from a noncoding 611-bp region. A 521-bp fragment contained within the 611-bp intergenic region could act as an origin of replication in Lactococcus lactis. Three major structural proteins, with sizes of 175, 90, and 29 kDa, and eight minor proteins, with sizes of 143, 82, 66, 60, 44, 42, 32, and 28 kDa, were identified. Several of these proteins appeared to be posttranslationally modified by proteolytic cleavage. The 175- and 90-kDa proteins were identified as the major phage head proteins, and the 29- and 60-kDa proteins were identified as the major tail protein and (possibly) the tail adsorption protein, respectively. The head proteins appeared to be covalently linked multimers of the same 30-kDa gene product. Phage c2 and prolate-headed lactococcal phage bIL67 (C. Schouler, S. D. Ehrlich, and M.-C. Chopin, Microbiology 140:3061-3069, 1994) shared 80% nucleotide sequence identity. However, several DNA deletions or insertions which corresponded to the loss or acquisition of specific ORFs, respectively, were noted. The identification of direct nucleotide repeats flanking these sequences indicated that recombination may be important in the evolution of these phages.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotiana benthamiana Plants Transformed with the 54-kDa Region of the Pepper Mild Mottle Tobamovirus Replicase Gene Exhibit Two Types of Resistance Responses against Vital Infection

Nicotiana benthamiana plants transformed with the 54-kDa region of the pepper mild mottle tobamovirus (PMMV) replicase gene were generated and six independently transformed plant lines were analyzed for resistance to PMMV. Two different resistance responses were obtained. Some of the transgenic plants from only two lines showed a preestablished, complete, and highly resistant phenotype since no vital symptoms were observed, although a low level of virus replication occurred The remaining plants from these two lines and all of the plants from the other four lines tested showed a delayed, induced, and also highly resistant phenotype since they were susceptible early, but were able to recover from the systemic PMMV infection. Recovered, symptomless leaves were resistant to the PMMV strain from which the 54-kDa gene was derived and to a closely related strain but not to tobacco mosaic virus. Such a delayed resistance phenotype has not been previously described for any plant expressing viral replicase sequences. The transgenic plants within the lines displaying complete or delayed resistance phenotypes were analyzed for transgene expression before and after PMMV inoculation and the two types of resistance responses were shown to be independent of the transgene transcript level.

Modified resistance of Solanum brevidens to potato Y potyvirus and tobacco mosaic tobamovirus following genetic transformation and explant regeneration

Infected plants of Solanum brevidens accumulate extremely low titres of potato Y potyvirus (PVY) and moderate titres of tobacco mosaic tobamovirus (TMV); but, in plants infected with both viruses, accumulation of PVY is enhanced, suggesting that TMV assists the cell-to-cell spread or replication of PVY. Cell-to-cell movement of TMV is mediated by the virus-coded 30-kDa movement protein. In this study, S. brevidens was transformed with the 30-kDa gene of TMV-204. Systemic spread of the temperature-sensitive mutant TMV-Ls1 occurred in four transgenic lines carrying the 30-kDa gene sense construct at the non-permissive temperature (33°C) indicating expression of functional wild type 30-kDa protein capable of complementing defective movement functions of TMV-Ls1. PVY accumulation was enhanced in three of these lines, in four transgenic ‘sense’ lines in which no complementation of TMV-Ls1 occurred, in nine ‘antisense’ lines, two lines lacking the 30-kDa gene insert, and nine non-transgenic callus-derived lines. No detectable infection or systemic spread of TMV-204 occurred in most of the callus-derived lines. Thus, enhanced accumulation of PVY and increased resistance to TMV appeared to be associated with tissue culture-induced changes rather than the 30-kDa gene insert.

VMA13 encodes a 54-kDa vacuolar H(+)-ATPase subunit required for activity but not assembly of the enzyme complex in Saccharomyces cerevisiae.

Previous purifications and characterizations of the Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) have indicated that this enzyme is a multisubunit complex composed of at least eight subunits of 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We report the cloning and characterization of an additional V-ATPase subunit, the 54-kDa subunit, which is encoded by the VMA13 gene. VMA13 was isolated by complementation of the growth phenotypes associated with the vma13 mutation, which was originally described as cls11 (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The nucleotide sequence of the VMA13 gene predicted a hydrophilic polypeptide with a calculated molecular mass of 54,415 daltons. The VMA13 54-kDa gene product resides on the vacuolar membrane and co-purified with the active V-ATPase complex. Characterization of a null vma13 mutant (delta vma13) revealed that the Vma13 polypeptide is essential for V-ATPase activity. However, the Vma13 polypeptide is not required for targeting of the other V-ATPase subunits (100-, 69-, 60-, 42-, 27-, or 17-kDa subunits) to the vacuolar membrane as shown by the association of these subunits with vacuolar membranes isolated from delta vma13 cells. The nature of the V-ATPase "complex" in delta vma13 mutant is, nevertheless, fundamentally different from the wild-type enzyme. This is evidenced by the fact that the inactive V-ATPase complex from delta vma13 cells is less stable than the wild-type enzyme. Taken together, these results indicate that VMA13 encodes the 54-kDa subunit of the V-ATPase and that this subunit is essential for activity, but not assembly, of the enzyme complex.

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.

A hybrid plant RNA virus made by transferring the noncapsid movement protein from a rod-shaped to an icosahedral virus is competent for systemic infection.

For many plant RNA viruses, multiple viral gene products, including noncapsid movement proteins and capsid proteins, contribute to the spread of infection within plants. The extent to which these factors interact to support infection spread is not known, but, for movement protein mutants of certain viruses, the inability of coinoculated "helper" viruses to complement defective movement has suggested a possible requirement for coadaptation between noncapsid movement proteins and other virus factors. To test directly for required coadaptation, the 3a movement protein gene of cowpea chlorotic mottle virus, an icosahedral bromovirus, was replaced with the nonhomologous 30-kDa movement protein gene of sunn-hemp mosaic virus, a rod-shaped, cowpea-adapted tobamovirus. The resulting hybrid virus is competent for systemic infection of cowpea, with systemic infection dependent upon expression of the 30-kDa gene. In view of the dramatic differences between cowpea chlorotic mottle virus and sunn-hemp mosaic virus in genetic organization and particle morphology, the ability of the hybrid to systemically infect cowpea implies that the tobamovirus 30-kDa movement protein functions independently of sequence-specific interactions with other viral components or sequences. Similarly, the required contribution of bromovirus capsid protein to infection movement appears to be independent of specific interaction with the natural 3a movement protein. In addition to other implications concerning movement protein and coat protein function, the results are consistent with the possibility that two or more distinguishable transfer processes may be involved in crossing different tissue barriers to achieve full systemic spread of infection.

Resistance to tobacco mosaic virus induced by the 54-kDa gene sequence requires expression of the 54-kDa protein.

Tobacco plants transformed with the sequence encoding the 54-kDa putative replicase protein of tobacco mosaic virus were resistant to systemic virus disease (D. B. Golemboski, G. P. Lomonossoff, and M. Zaitlin, Proc. Natl. Acad. Sci. USA 87:6311-6315, 1990). Resistance was due to a marked suppression of virus replication at the site of inoculation (J. P. Carr and M. Zaitlin, Mol. Plant-Microbe Interact. 4:579-585, 1991). Although RNA transcripts encoding the 54-kDa protein were present in resistant plants, the 54-kDa protein itself was not observed in vivo. We wished to assess the relative importance of the 54-kDa protein versus its RNA in mediating resistance. Further attempts to detect the 54-kDa protein in plant tissues were unsuccessful; therefore, an indirect approach was taken using a protoplast-based transient gene expression system. Electroporation of protoplasts with plasmids capable of expressing the wild-type 54-kDa protein gene sequence or a mutant lacking the first AUG initiation codon of the 54-kDa open reading frame and encoding a slightly truncated protein reduced virus replication in protoplasts. In contrast, a frameshift mutant that was capable of directing synthesis of a protein only 20% the size of the 54-kDa protein, did not produce resistance in protoplasts. These results show that expression of the 54-kDa protein gene sequence at the RNA level alone is insufficient for resistance, and they implicate the 54-kDa protein itself in mediating this resistance phenomenon.

Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus.

Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 micrograms/ml or 300 micrograms/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 micrograms/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to approximately 5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected.

In vitro transcription and translation of cloned cDNAs encoding the 30-kDa protein gene of TMV

A cDNA clone encoding the nonstructural, 30-kDa protein of the common (Ul) strain of tobacco mosaic virus (TMV) was isolated and characterized. cDNA clones representing the intact gene as well as deletions from the 5′ end of the gene were subcloned into SP6 vectors. Capped RNAs produced by in vitro transcription reactions were translated in a wheat germ cell-free system. The resultant proteins were compared to proteins obtained from the in vitro translation of intermediate length (12) rods of TMV. Transcripts of the cDNA clones encoded polypeptides of 30, 28, or 18 kDa that were immunoprecipitated by antibody prepared against a synthetic peptide representing the carboxy terminus of the 30-kDa protein. eDNA clones containing the intact 30-kDa sequence coded for 30-kDa polypeptides while clones lacking the 30-kDa initiation codon produced 28-kDa polypeptides. Surprisingly, translation of a transcript from a cDNA clone containing the 30-kDa gene plus 390 nucleotides 5′ of the initiator AUG yielded a polypeptide with an approximate molecular mass of 18 kDa. The results indicate that an intact and functional 30-kDa protein gene has been cloned. The significance of these results, with respect to determining the function of the 30-kDa protein, is discussed.

Cotranslational disassembly of a cowpea strain ( Cc) of TMV: Evidence that viral RNA-protein interactions at the assembly origin block ribosome translocation in vitro

Cotranslational disassembly of mixed rod-length populations of tobacco mosaic virus (TMV; vulgare, common or, U1 strain), reveals a reproducible, significant reduction in the level of expression of the intermediate-sized, subgenomic 12-RNA when compared with conventional total viral RNA preparations. I 2-RNA encodes a 30-kDa protein, and recent evidence suggests that I 2-RNA is unusual in that it lacks a 5′-cap structure. In TMV vulgare, the assembly origin is located within the 30-kDa protein-coding region. To resolve which of these structural features might be responsible for the decline in 30-kDa gene expression from packaged 12-RNAs, the products encoded in vitro by packaged or naked genomic and subgenomic RNAs from two strains of TMV, vulgare and a cowpea strain ( Cc or Sunn-hemp mosaic virus), were compared. The results indicate that strong coat protein-RNA interactions, presumed to occur at the assembly origin, dictate the site at which translocation of 80 S ribosomes is inhibited. The implications of this conclusion for virus infection in vivo are discussed.