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In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries
MONITOR
A 'digest' of some recent papers of interest in the primary journals.
A P-glyc0protein protects Caenorbabditis elegans against natural toxins
In situ isohtion of mRNAfromindividual plantcells:creationof cell-specific cDNAlibraries
A. BROEKS, H.W.R.M. JANSSEN, J. CALAFAT AND R.H.A. PLASTERK
E.E. KARRER ETAL.
Proc. Natl Acad. Sci. USA 92,
~ B O J . 14, 1858-1866
3814-3818
Drug resistance mediated by P-glycoproteins is a serious clinical problem in controlling cancer and in combating parasitic diseases. Cells as different as mammalian turnout cells and protozoa are able to increase resistance to a variety of cytotoxic drugs by increasing the level of these transport proteins, usually by gene amplification. However, drug transport is not the only function of this gene family, because one of the two human genes, MDR3, is involved in phosphatidyl choline secretion, not in drag transport. Broeks et al. have investigated P-glycoproteins in Caenorhabditis elegans, a non-parasitic soil nematode with a lifestyle very different from a mammal or a protozoan parasite. C. elegans has four P-glycoprotein genes. Two of these, pgp-1 arid PgP-3, have been deleted by transposon-excision, an increasingly efficient technique in C. elegans research. The PgP-3 mutant does indeed show a marked increase in sensitivity to colchicine and chloroquine, but the pgp-1 mutant does not, nor is there any further increase in sensitivity in the double mutant. Increased copy number of the pgp genes does not increase resistance, perhaps because the drug-exporting system is already working at a maximal level. Expression studies indicate that both genes are expressed in the intestinal cells, but that PgP-3 is also expressed in the excretory cell, a large cell that probably acts as the nematode kidney. This is consistent with the proposed role of PgP-3 as a defence against plant toxins encountered in the natural environment. The pgp-lmutant has no discernible defects, so the functions of this gene, and of pgp-2 and pgp-4, remain mysterious, a:a
In multicellular organisms, cells are differentiated from their neighbours by their patterns of gene expression. Current methods for examining these differences have relied on in situ hybridization, or PCR using primers designed to detect a specific transcript. The disadvantages of these techniques are that fixed tissue must be used and that only the presence of a known InRNA sequence can be analysed. This paper provides a new approach. Using micropipettes and intact plant tissue, the contents of a single cell were aspirated. RNA was extracted and mRNA purified on
Herpessimplexvirusturnsoff the TAPto evadehost immunity A. HILL ETAL.
Nature 375, 411-415 The recognition and destruction of virus-infected cells mediated by MHC class I-restricted cytotoxic T lymphocytes (CTL) is a major factor in establishing immune control of viral infections. Currently, four viruses are known to evade host CTL responses by interfering post-translationally with MHC class I-restricted antigen presentation: adenovims; human and routine cytomegalovims; and herpes simplex virus. The interactions of the adenovirus E3 protein with MHC class I and subsequent retention of HLA molecules in the ER, have been well studied, but the precise mechanisms by which the other viruses affect antigenprocessing have not been well understood. Previously, these authors have
oligo(dT)-linked magnetic beads. After cDNA synthesis and addition of adaptors, the cDNA was amplified by PCR and used to construct a library. The specificity of the method has been verified by showing that stomatal guard cells contained a proton ATPase mRNA, whereas the neighbouring leaf epidermal cells did not. As the authors acknowledge, the method is not yet perfect; rare transcripts are sporadically represented in the final cDNA library, as each of the initial cDNA templates has a certain probability of being amplified or of being lost (a phenomenon the authors dub the 'Monte Carlo Effect'). Nevertheless, it seems likely that further advances will improve on the method and molecular biology will have a powerful new tool with which to study gene activity. All it needs is the ability to think and manipulate really small. /~
shown that HSV infection of human fibroblasts results in a failure of class I molecules to assemble and move to the cell surface, an effect mediated by an HSV protein named ICP47. They now demonstrate that ICP47 binds to the transporter associated with antigen processing (TAP), thus blocking peptide translocation from the cytosol to the ER. In the absence of peptide, class I molecules fail to fold correctly and are retained and degraded in the ER. Studies such as this, shed light on the nature of the virus-host relationship; avoidance of CTL recognition might create the opportunity for HSV to replicate and achieve latency before the immune system can establish effective control of the infection. Further, herpesvimses have probably been coevolving with humans for millions of years and so it is interesting to speculate that further study of these viruses may illuminate other aspects of immune physiology.
It's e a s y t o s u b s c r i b e t o T / G Just email or call or fax
[email protected] +44 1865 843300 or +1 914 524 9200 +44 1865 843940 or + i 9 1 4 333 2k-~{
All you need to provide is your name, address and the month from which you would like your subscription to start, your credit card number and its expiry date. (Please do not send credit card details by email.)
301
A 'digest' of some recent papers of interest in the primary journals.
A P-glyc0protein protects Caenorbabditis elegans against natural toxins
In situ isohtion of mRNAfromindividual plantcells:creationof cell-specific cDNAlibraries
A. BROEKS, H.W.R.M. JANSSEN, J. CALAFAT AND R.H.A. PLASTERK
E.E. KARRER ETAL.
Proc. Natl Acad. Sci. USA 92,
~ B O J . 14, 1858-1866
3814-3818
Drug resistance mediated by P-glycoproteins is a serious clinical problem in controlling cancer and in combating parasitic diseases. Cells as different as mammalian turnout cells and protozoa are able to increase resistance to a variety of cytotoxic drugs by increasing the level of these transport proteins, usually by gene amplification. However, drug transport is not the only function of this gene family, because one of the two human genes, MDR3, is involved in phosphatidyl choline secretion, not in drag transport. Broeks et al. have investigated P-glycoproteins in Caenorhabditis elegans, a non-parasitic soil nematode with a lifestyle very different from a mammal or a protozoan parasite. C. elegans has four P-glycoprotein genes. Two of these, pgp-1 arid PgP-3, have been deleted by transposon-excision, an increasingly efficient technique in C. elegans research. The PgP-3 mutant does indeed show a marked increase in sensitivity to colchicine and chloroquine, but the pgp-1 mutant does not, nor is there any further increase in sensitivity in the double mutant. Increased copy number of the pgp genes does not increase resistance, perhaps because the drug-exporting system is already working at a maximal level. Expression studies indicate that both genes are expressed in the intestinal cells, but that PgP-3 is also expressed in the excretory cell, a large cell that probably acts as the nematode kidney. This is consistent with the proposed role of PgP-3 as a defence against plant toxins encountered in the natural environment. The pgp-lmutant has no discernible defects, so the functions of this gene, and of pgp-2 and pgp-4, remain mysterious, a:a
In multicellular organisms, cells are differentiated from their neighbours by their patterns of gene expression. Current methods for examining these differences have relied on in situ hybridization, or PCR using primers designed to detect a specific transcript. The disadvantages of these techniques are that fixed tissue must be used and that only the presence of a known InRNA sequence can be analysed. This paper provides a new approach. Using micropipettes and intact plant tissue, the contents of a single cell were aspirated. RNA was extracted and mRNA purified on
Herpessimplexvirusturnsoff the TAPto evadehost immunity A. HILL ETAL.
Nature 375, 411-415 The recognition and destruction of virus-infected cells mediated by MHC class I-restricted cytotoxic T lymphocytes (CTL) is a major factor in establishing immune control of viral infections. Currently, four viruses are known to evade host CTL responses by interfering post-translationally with MHC class I-restricted antigen presentation: adenovims; human and routine cytomegalovims; and herpes simplex virus. The interactions of the adenovirus E3 protein with MHC class I and subsequent retention of HLA molecules in the ER, have been well studied, but the precise mechanisms by which the other viruses affect antigenprocessing have not been well understood. Previously, these authors have
oligo(dT)-linked magnetic beads. After cDNA synthesis and addition of adaptors, the cDNA was amplified by PCR and used to construct a library. The specificity of the method has been verified by showing that stomatal guard cells contained a proton ATPase mRNA, whereas the neighbouring leaf epidermal cells did not. As the authors acknowledge, the method is not yet perfect; rare transcripts are sporadically represented in the final cDNA library, as each of the initial cDNA templates has a certain probability of being amplified or of being lost (a phenomenon the authors dub the 'Monte Carlo Effect'). Nevertheless, it seems likely that further advances will improve on the method and molecular biology will have a powerful new tool with which to study gene activity. All it needs is the ability to think and manipulate really small. /~
shown that HSV infection of human fibroblasts results in a failure of class I molecules to assemble and move to the cell surface, an effect mediated by an HSV protein named ICP47. They now demonstrate that ICP47 binds to the transporter associated with antigen processing (TAP), thus blocking peptide translocation from the cytosol to the ER. In the absence of peptide, class I molecules fail to fold correctly and are retained and degraded in the ER. Studies such as this, shed light on the nature of the virus-host relationship; avoidance of CTL recognition might create the opportunity for HSV to replicate and achieve latency before the immune system can establish effective control of the infection. Further, herpesvimses have probably been coevolving with humans for millions of years and so it is interesting to speculate that further study of these viruses may illuminate other aspects of immune physiology.
It's e a s y t o s u b s c r i b e t o T / G Just email or call or fax
[email protected] +44 1865 843300 or +1 914 524 9200 +44 1865 843940 or + i 9 1 4 333 2k-~{
All you need to provide is your name, address and the month from which you would like your subscription to start, your credit card number and its expiry date. (Please do not send credit card details by email.)
301