BACKGROUND
potato virus x , a potexvirus, is a filamentous rod-shaped virus which contains a single plus-sense rna molecule.
the rna is capped, polyadenylated, and encodes five open reading frames  <cit> .
pvx was designed to express foreign genes <cit> .
its coat promoter was duplicated in the vector to drive the foreign orf transcription.
the pvx vector has been used to express foreign genes in many host plants <cit> , in translation studies <cit> , and for investigating gene silencing <cit> .
in the current study, the pvx vector was used to express the gp <dig> gene of bvdv singer strain <cit>  and vp <dig> of cpv- <dig> <cit>  for vaccine development in a plant vector.
the construction of the pvx vector required the new plasmid to be amplified in e. coli before inoculating plants, which would be necessary to construct a plant virus vectored viral genes for a vaccine for these two viruses.
in molecular cloning, plasmids need to be amplified in e. coli to produce enough plasmid dna for molecular manipulation.
the foreign gene in the expression vectors is not expressed until it is in a eukaryotic host cell; thus, it was surprising to find, when the plasmid was amplified in e. coli, the foreign gene was expressed and it killed the bacteria.
cryptic promoter is the promoter homologous sequence that distribute randomly.
it was previously reported that there was a cryptic promoter associated with a toxic gene in potato virus y  that interrupted cloning work in e. coli <cit> .
the cryptic promoter in pvx has not been reported before.
when the eukaryotic expression vectors are derived from viruses, certain viral sequences might be recognized as promoter elements.
we tried to sub-clone two animal viral genes into the pvx vector; however, only the gp <dig> orf from bvdv was successfully sub-cloned.
the other gene, the cpv- <dig> vp <dig>  could not be sub-cloned into the pvx vector, probably because it was toxic to e. coli when expressed.
if vp <dig> had not been expressed in e. coli, the cloning would have been possible and the cryptic promoter in the pvx vector would not have been found, as occurred when the gp <dig> orf was sub-cloned in the pvx vector and transcribed.
the transcription initiation site was determined with rt-pcr.
the cryptic promoter activity from pvx was compared with that of the t <dig> promoter with real-time rt-pcr.
RESULTS
comparison of the pvx cryptic promoter with the conserved prokaryotic promoter
the similarity of the end element of the triple block sequences in pvx and the prokaryotic conserved sequence of - <dig> and - <dig> were compared .
the conserved promoter sequences of prokaryotic organisms are - <dig> tataat and - <dig> ttgaca, separated by 16– <dig> base pairs.
the prokaryotic homologous element in the pvx vector matched over 50% of the prokaryotic conserved promoter sequence.
the homologous element was considered as the cryptic promoter sequence and it was hypothesized to drive the transcription of foreign genes.
transcription of foreign genes driven by the cryptic promoter on the pvx vector
both the bvdv gp <dig> and the cvp- <dig> vp <dig> foreign genes were sub-cloned into the pvx vector.
however, only the gp <dig> orf in the plasmid could be expressed.
in the pvx vector, the cloned vp <dig> fragment always had a mutation near the start codon .
we hypothesized that there was a cryptic promoter driving the foreign gene to be transcribed, which lead to expression of a toxic vp <dig> protein that killed the e. coli during plasmid amplification.
in order to demonstrate the existence of cryptic promoter, e. coli was transformed with the pvx vector, pgr106gp <dig> plasmid.
after a four hour culture, the total rna was isolated.
northern blots showed the foreign gene gp <dig> was transcribed .
with the gp <dig> specific probe in northern blots, the pgr106gp <dig> transformed e. coli showing a band of about  <dig> kb; whereas, the plasmid pgr106-transformed e. coli  did not.
this suggests that cryptic promoter activity existed.
determination of the site of transcription initiation
if the hypothesis regarding the cryptic promoter position was correct , the gp <dig> rna in e. coli should have a 5' untranslated region  from the pvx vector.
based on the sequence behind the cryptic promoter, three different primers in front of the gp <dig> orf  and an antisense primer from gp <dig> sequence  were designed to run rt-pcr.
only primer  <dig>  could amplify the cdna of gp <dig>  the 5'utr is about  <dig> base pairs long .
a negative control plasmid  transformed e. coli, did not have any pcr product, using gp <dig> specific primer.
the mrna control  showed that the rna was not contaminated with plasmid dna because no dna fragment was amplified.
this demonstrated that transcription was initiated at approximately  <dig> base pairs downstream of the cryptic promoter.
comparison of the cryptic promoter with the t <dig> promoter using real-time rt-pcr
the activity of both the cryptic promoter and t <dig> promoter  were compared using real-time rt-pcr.
this method uses mathematical formulas to calculate relative expression levels compared to a calibrator.
the amount of target gene expression is also normalized to an endogenous housekeeping gene, in this case, 16s rrna.
the cryptic promoter activity was significantly higher than that of the t <dig> promoter  .
CONCLUSIONS
even though the cryptic promoter sequence did not perfectly match the conserved prokaryotic - <dig> and - <dig> elements, it matched 50% and 66%, respectively .
it has been reported that certain foreign elements can be recognized as a promoter by e. coli <cit> .
promoter homologous sequences are widely distributed in the genomic sequence of e. coli <cit> .
cryptic promoters are those sequences that are promoter homologous sequences driving transcription.
the cryptic sequences might also interrupt expressions of foreign genes in plants.
an example is that the homologous intron sequence within a gene can also interrupt expression of foreign gene <cit> .
in the current study, a prokaryotic cryptic promoter drove transcription during plasmid amplification.
the sequence of the cryptic promoter in pvx is different from that in pvy <cit> .
the interruption was different because the toxic gene is not from the virus itself as in pvy but from the foreign gene.
cryptic promoters in eukaryotic cells can have different functions, such as increased enzyme expression; tissue-specific or developmental stage-specific gene expression  <cit> .
a reporter gene, such as chloramphenicol acetyltransferase  when sub-cloned behind different promoters could be used to compare the promoter activity <cit> .
the enzyme expression driven by promoters can reflect the promoters' activities.
however, with real-time rt-pcr, the transcribed rna could be used as an indicator to show the promoter activity <cit> .
with real time rt-pcr, the cryptic promoter activity was compared with that of t <dig> promoter, an inducible promoter.
although the pvx vector is an expression vector in plants, it needs to be amplified in e. coli for molecular manipulation.
during the dna amplifying stage, the foreign genes should not be expressed; however, the foreign genes in the pvx vector were transcribed.
if vp <dig> were not toxic to bacteria its transcription would not influence the cloning work.
the failure to sub-clone the vp <dig> cistron, suggested that vp <dig> was toxic to bacteria .
the toxic pressure prevented the non-mutated vp <dig> from being sub-cloned into the pvx vector.
a number of plant viruses, such as tobacco mosaic virus  and pvx, have been reported to contain mrna regions that possess a high degree of homology to both chloroplast rrna and prokaryotic 16s rna, which are closely related <cit> .
it has been demonstrated that a triple block sequence located upstream from the coat protein gene of pvx facilitated the translation of the pokeweed antiviral protein gene in e. coli  <cit> .
even though unexpected expression of a foreign gene can interrupt downstream cloning work, the cryptic promoter makes the pvx vector an expression vector in e. coli, able to express foreign genes in both e. coli and plants.
this ability to express foreign genes either in a prokaryotic organism  or a eukaryotic organism  makes the pvx vector a potential useful tool to study the differences in transcription and translation between prokaryotic and eukaryotic organisms without changing the vector.

 potato virus x, a potexvirus, is a filamentous rod-shaped virus which contains a single plus-sense rna molecule. the rna is capped, polyadenylated, and encodes five open reading frames. pvx was designed to express foreign genes. its coat promoter was duplicated in the vector to drive the foreign orf transcription. the pvx vector has been used to express foreign genes in many host plants, in translation studies, and for investigating gene silencing. in the current study, the pvx vector was used to express the gp gene of bvdv singer strain and vp of cpv- for vaccine development in a plant vector. the construction of the pvx vector required the new plasmid to be amplified in e. coli before inoculating plants, which would be necessary to construct a plant virus vectored viral genes for a vaccine for these two viruses. in molecular cloning, plasmids need to be amplified in e. coli to produce enough plasmid dna for molecular manipulation. the foreign gene in the expression vectors is not expressed until it is in a eukaryotic host cell; thus, it was surprising to find, when the plasmid was amplified in e. coli, the foreign gene was expressed and it killed the bacteria. cryptic promoter is the promoter homologous sequence that distribute randomly. it was previously reported that there was a cryptic promoter associated with a toxic gene in potato virus y that interrupted cloning work in e. coli. the cryptic promoter in pvx has not been reported before. when the eukaryotic expression vectors are derived from viruses, certain viral sequences might be recognized as promoter elements. we tried to sub-clone two animal viral genes into the pvx vector; however, only the gp orf from bvdv was successfully sub-cloned. the other gene, the cpv- vp could not be sub-cloned into the pvx vector, probably because it was toxic to e. coli when expressed. if vp had not been expressed in e. coli, the cloning would have been possible and the cryptic promoter in the pvx vector would not have been found, as occurred when the gp orf was sub-cloned in the pvx vector and transcribed. the transcription initiation site was determined with rt-pcr. the cryptic promoter activity from pvx was compared with that of the t promoter with real-time rt-pcr.  comparison of the pvx cryptic promoter with the conserved prokaryotic promoter the similarity of the end element of the triple block sequences in pvx and the prokaryotic conserved sequence of - and - were compared. the conserved promoter sequences of prokaryotic organisms are - tataat and - ttgaca, separated by 16– base pairs. the prokaryotic homologous element in the pvx vector matched over 50% of the prokaryotic conserved promoter sequence. the homologous element was considered as the cryptic promoter sequence and it was hypothesized to drive the transcription of foreign genes. transcription of foreign genes driven by the cryptic promoter on the pvx vector both the bvdv gp and the cvp- vp foreign genes were sub-cloned into the pvx vector. however, only the gp orf in the plasmid could be expressed. in the pvx vector, the cloned vp fragment always had a mutation near the start codon. we hypothesized that there was a cryptic promoter driving the foreign gene to be transcribed, which lead to expression of a toxic vp protein that killed the e. coli during plasmid amplification. in order to demonstrate the existence of cryptic promoter, e. coli was transformed with the pvx vector, pgr106gp plasmid. after a four hour culture, the total rna was isolated. northern blots showed the foreign gene gp was transcribed. with the gp specific probe in northern blots, the pgr106gp transformed e. coli showing a band of about kb; whereas, the plasmid pgr106-transformed e. coli did not. this suggests that cryptic promoter activity existed. determination of the site of transcription initiation if the hypothesis regarding the cryptic promoter position was correct, the gp rna in e. coli should have a 5' untranslated region from the pvx vector. based on the sequence behind the cryptic promoter, three different primers in front of the gp orf and an antisense primer from gp sequence were designed to run rt-pcr. only primer could amplify the cdna of gp the 5'utr is about base pairs long. a negative control plasmid transformed e. coli, did not have any pcr product, using gp specific primer. the mrna control showed that the rna was not contaminated with plasmid dna because no dna fragment was amplified. this demonstrated that transcription was initiated at approximately base pairs downstream of the cryptic promoter. comparison of the cryptic promoter with the t promoter using real-time rt-pcr the activity of both the cryptic promoter and t promoter were compared using real-time rt-pcr. this method uses mathematical formulas to calculate relative expression levels compared to a calibrator. the amount of target gene expression is also normalized to an endogenous housekeeping gene, in this case, 16s rrna. the cryptic promoter activity was significantly higher than that of the t promoter.  even though the cryptic promoter sequence did not perfectly match the conserved prokaryotic - and - elements, it matched 50% and 66%, respectively. it has been reported that certain foreign elements can be recognized as a promoter by e. coli. promoter homologous sequences are widely distributed in the genomic sequence of e. coli. cryptic promoters are those sequences that are promoter homologous sequences driving transcription. the cryptic sequences might also interrupt expressions of foreign genes in plants. an example is that the homologous intron sequence within a gene can also interrupt expression of foreign gene. in the current study, a prokaryotic cryptic promoter drove transcription during plasmid amplification. the sequence of the cryptic promoter in pvx is different from that in pvy. the interruption was different because the toxic gene is not from the virus itself as in pvy but from the foreign gene. cryptic promoters in eukaryotic cells can have different functions, such as increased enzyme expression; tissue-specific or developmental stage-specific gene expression. a reporter gene, such as chloramphenicol acetyltransferase when sub-cloned behind different promoters could be used to compare the promoter activity. the enzyme expression driven by promoters can reflect the promoters' activities. however, with real-time rt-pcr, the transcribed rna could be used as an indicator to show the promoter activity. with real time rt-pcr, the cryptic promoter activity was compared with that of t promoter, an inducible promoter. although the pvx vector is an expression vector in plants, it needs to be amplified in e. coli for molecular manipulation. during the dna amplifying stage, the foreign genes should not be expressed; however, the foreign genes in the pvx vector were transcribed. if vp were not toxic to bacteria its transcription would not influence the cloning work. the failure to sub-clone the vp cistron, suggested that vp was toxic to bacteria. the toxic pressure prevented the non-mutated vp from being sub-cloned into the pvx vector. a number of plant viruses, such as tobacco mosaic virus and pvx, have been reported to contain mrna regions that possess a high degree of homology to both chloroplast rrna and prokaryotic 16s rna, which are closely related. it has been demonstrated that a triple block sequence located upstream from the coat protein gene of pvx facilitated the translation of the pokeweed antiviral protein gene in e. coli. even though unexpected expression of a foreign gene can interrupt downstream cloning work, the cryptic promoter makes the pvx vector an expression vector in e. coli, able to express foreign genes in both e. coli and plants. this ability to express foreign genes either in a prokaryotic organism or a eukaryotic organism makes the pvx vector a potential useful tool to study the differences in transcription and translation between prokaryotic and eukaryotic organisms without changing the vector. 
a cryptic promoter in the pvx vector was discovered with rt-pcr. it is widely used to express foreign genes. in molecular manipulation, the foreign genes need to be sub-cloned into the vector. usually, during amplification stage, the foreign genes are not expressed. potato virus x has been developed into an expression vector for plants. however, if the foreign gene is expressed, the construction work could be interrupted. two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. the other foreign gene, canine parvovirus type vp could not be sub-cloned into the vector and amplified without mutation. it is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. the promoter activity was studied with northern blots and real-time rt-pcr. during the plasmid amplification stage, an unexpected expression of the cpv- vp gene can interrupt the downstream work.
 potato virus x has been developed into an expression vector for plants. it is widely used to express foreign genes. in molecular manipulation, the foreign genes need to be sub-cloned into the vector. the constructed plasmid needs to be amplified. usually, during amplification stage, the foreign genes are not expressed. however, if the foreign gene is expressed, the construction work could be interrupted. two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. the other foreign gene, canine parvovirus type vp could not be sub-cloned into the vector and amplified without mutation.  a cryptic promoter in the pvx vector was discovered with rt-pcr. the promoter activity was studied with northern blots and real-time rt-pcr.  it is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. during the plasmid amplification stage, an unexpected expression of the cpv- vp gene can interrupt the downstream work. 