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Activity of viral promotors in cultered human skin cells
Poster Presentations
85
429
432 ACIIVITV CELLS.-
VIRAL PROMOTORS IN CULTERED HUMAN SKIN Arfuc. Wolf Nlmbere. Matthias Platur*. Beats A nd Dirk Schaden_d& UKRV, Dermatology, and MaxDellbriick-Center for Molecular Medicine(*), FU Berlin Promotor and enhancer activities that control gene transcription vary considerably among different cell types. The combination of different recognition sequences and the amounts of cognate transcription factors determine the efficiency with which a given gene is transcribed in a particular cell type. In order to develop systems to transiently or stablly express mammalian proteins in human skin-derived cells, we tested 7 different viral promotors (pCMV, pRSV, pHMT, pPMTV, PUPE, PUPL, pSVE) to drive the expression of the chlorampheoicol acetyltransferase (CAT) enzyme in cpithelial cetls (HT.3), human normaI keratinocytes and neuroectodermal cells (SK-Mel 7.3, SK-Mel 37). DNA was transfected using a liposome-based technique and transfection efficacy was cootrolled by cotraosfectioa of a Bgalactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined by incubation of the cell extract prepared from transferted cells with 14C-labeled chloramphenicol. CAT activity was correlated to the S-galactosidase activity and protein amount. In conclusion. strategies for overexpression of foreign genes in human epithelial and melaaocytic cells should consider the usage of the CMV promotor, since only the viral replicon of CMV and less so of RSV were active in our cell-line tested. The expression plasmid containing the SVE promoter was only active on the epithel cell-lines tested and seems to be of sufficiently value m studinggene expression in human epidcrmal cells.
430
433
NU-[DEDIPHOSPHArr KWASEEXRtESSIONINHClMANSKIN. w Depanment of Dermatology. School of Medicine. i..
XIc&dh9 The Univcrsily
of Tokoshima Tokurhima. Japan Recent smdics have damonswarcd ttx prosonce of "ucleoride diphosphare (NDP) kinsse in assosiatio” with protems presenting sect speclficiry for geanine nucleotides, including microlubules, initiation facmr eE?. and G proteins, suggesting that NDP kinase may play a role in many cellular events by reple”irbme”t of “ucleoside triphospharcs (NTPs) in the immediate vicinw of the NTP-requiring reactions. Few smdies on NDP kinase expression m human ski” have bee” performed so far. In this rrudy. we examined NDP kinase cxpreswo” in human skin using a rabbit poly~lonal anlibody against ral liver NDP kinase. A” mmwnoblot analysis using whole skm ext~aels delecled a 19 kDa band. 11s ,mm”“ohis,ocbem,cal size corresponded to that of ral liver NDP kmase. analyses showed the presence of NDP kinare a” the epldennis. haar follicles. and cccrine and apocrine swear glands f” the epudermis. basal and suprabasal cells I” ,he o”,er root *heath Of a were more *lro”gly posilive than spmous cells ba,r ,o,,,c,e. Ihe outermos, layer was more deeply slained than ,nner layers. In the eccrine sweat duct, basal ductal cells were more strongly positive than luminal cells. These findings suggest thal NDP kinase II expressed more abundantly in membolically active cells of human ski”.
431 CALRET,C”LlN (CR, ISTRANSCR~PT~ONACL~REG”~~~~~ BYHEATSHOCK. QJ. Noqven.
J.D.
Ceora,
R.D.
Sontheimer.
Depts.
of Dermatology
and Microbiology.
U.T. Southwestern Med. Center. Dallas. TX. 75235. CR has recently been confirmed to be a new human rheumatic d&seas-associated autoantigen. In bur studies. thus 46 kD (SO kD in SDS-PAGE) hngh-affinity calciumbinding protein of unknown function IS phvs~cmllv assoaated wth the ROBS-A autoantlgen complex. Since cellular modulatmn of Ro/SS-A anngen expressiOn has been implicated I” the pathogenesls of subacute cutaneous lupus ervthematosus ILE) and neonatal LE. we have begun to examine the genetuz regulation of CR. To examine transcriptlonel regulation of CR. a reporter gene construct was developed bv msertino a 51 1 bo fraoment of the 5’ flsnkino reoion of a oenomic clone of CR & a- &&d co&i& a bacterial chloramphenicol ace~vltransferase (CAT1 reoorter wane (CR-CAT). CR-CAT was then transfected Into A431 cells and relect,ve-&wth ,n G418 was used to isolate three clones that were confIrmed bv polymerase cham reactionandSouthernblatting tohave CR-CA7sfablyintegrated intooenomlc DNA. Each of the three cloneswa~ to constitutively express
found
low
OF
avels
of CAT
activitv
as detected
bv thin
Iaver chromatowaohv.
Calcium
&ophore shock lAZ3lSi 2.5 @firnIl. heat shock l43OC x 1-W and Zinc ion exoosure (225 uMI were found to mcrease CAT expression in these clones bV SOOl.iOO%. 400.‘600% and 500.1.700% respect,ve,V. Studies are underwav to exam,ne the effects of “ltrav,olet B hght on CR-CAT express,on These reSults suggest that al the 51 fragment of the 5’ regulatory reg#on examined in this study 1s sufflclent for promoting transcrlptlon of the human CR gene, bl CR is const~tuwelv transcribed I” a transformed epidermal kerat8nocyte hne, c) CR is regulated at the transcrlptlonal level, and d) CR, like several other LE-related autoant,gens, appears to function as a heat shock gene
1bp
C’IVDYCF ZTEilOIL SULii,WBE GWE LiELETIONS ANI: CEATECTION OF THE WRRIWS &ilan Hu and Li Fang, Departiaent of Lernatology. tiua?%an iiospital, Shanghai Nedice. University, Shanghai, F.R.ihina Recessive X-linked Ichthyosis(RXI.1) is one of the nest common x-linked genetic disorders, which is attributed to deficiency of steroid sulfatase(STS). In approximatelv W% of the natients. this lack is due to the deletion of 53 gene-which is located at Xp22.3. The &.A probe of STS gene has been isolated previously by other cmups. For the first time in China, we analyzed 19 ca*e* of RALI ~IYJ 11 cases of Autosomal Lnminant IchthyosisjiLI) oy Southern olot hybridization and Polymense 3hain ReactloniPCR) as well. We found that 84% (16 out of 19) FXLI have STS gene deletions vhile kL1 haven't any. iurtnermore, as the carriers have half-normal gene dosage, we have used gene dosage analysis to detect the carriers in four RXLI families and found two "others be hetemzygote. These experiments contributed to rapid. simple and effective gene diagnosis and prenatal diagnosis of R.LI.
434
85
429
432 ACIIVITV CELLS.-
VIRAL PROMOTORS IN CULTERED HUMAN SKIN Arfuc. Wolf Nlmbere. Matthias Platur*. Beats A nd Dirk Schaden_d& UKRV, Dermatology, and MaxDellbriick-Center for Molecular Medicine(*), FU Berlin Promotor and enhancer activities that control gene transcription vary considerably among different cell types. The combination of different recognition sequences and the amounts of cognate transcription factors determine the efficiency with which a given gene is transcribed in a particular cell type. In order to develop systems to transiently or stablly express mammalian proteins in human skin-derived cells, we tested 7 different viral promotors (pCMV, pRSV, pHMT, pPMTV, PUPE, PUPL, pSVE) to drive the expression of the chlorampheoicol acetyltransferase (CAT) enzyme in cpithelial cetls (HT.3), human normaI keratinocytes and neuroectodermal cells (SK-Mel 7.3, SK-Mel 37). DNA was transfected using a liposome-based technique and transfection efficacy was cootrolled by cotraosfectioa of a Bgalactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined by incubation of the cell extract prepared from transferted cells with 14C-labeled chloramphenicol. CAT activity was correlated to the S-galactosidase activity and protein amount. In conclusion. strategies for overexpression of foreign genes in human epithelial and melaaocytic cells should consider the usage of the CMV promotor, since only the viral replicon of CMV and less so of RSV were active in our cell-line tested. The expression plasmid containing the SVE promoter was only active on the epithel cell-lines tested and seems to be of sufficiently value m studinggene expression in human epidcrmal cells.
430
433
NU-[DEDIPHOSPHArr KWASEEXRtESSIONINHClMANSKIN. w Depanment of Dermatology. School of Medicine. i..
XIc&dh9 The Univcrsily
of Tokoshima Tokurhima. Japan Recent smdics have damonswarcd ttx prosonce of "ucleoride diphosphare (NDP) kinsse in assosiatio” with protems presenting sect speclficiry for geanine nucleotides, including microlubules, initiation facmr eE?. and G proteins, suggesting that NDP kinase may play a role in many cellular events by reple”irbme”t of “ucleoside triphospharcs (NTPs) in the immediate vicinw of the NTP-requiring reactions. Few smdies on NDP kinase expression m human ski” have bee” performed so far. In this rrudy. we examined NDP kinase cxpreswo” in human skin using a rabbit poly~lonal anlibody against ral liver NDP kinase. A” mmwnoblot analysis using whole skm ext~aels delecled a 19 kDa band. 11s ,mm”“ohis,ocbem,cal size corresponded to that of ral liver NDP kmase. analyses showed the presence of NDP kinare a” the epldennis. haar follicles. and cccrine and apocrine swear glands f” the epudermis. basal and suprabasal cells I” ,he o”,er root *heath Of a were more *lro”gly posilive than spmous cells ba,r ,o,,,c,e. Ihe outermos, layer was more deeply slained than ,nner layers. In the eccrine sweat duct, basal ductal cells were more strongly positive than luminal cells. These findings suggest thal NDP kinase II expressed more abundantly in membolically active cells of human ski”.
431 CALRET,C”LlN (CR, ISTRANSCR~PT~ONACL~REG”~~~~~ BYHEATSHOCK. QJ. Noqven.
J.D.
Ceora,
R.D.
Sontheimer.
Depts.
of Dermatology
and Microbiology.
U.T. Southwestern Med. Center. Dallas. TX. 75235. CR has recently been confirmed to be a new human rheumatic d&seas-associated autoantigen. In bur studies. thus 46 kD (SO kD in SDS-PAGE) hngh-affinity calciumbinding protein of unknown function IS phvs~cmllv assoaated wth the ROBS-A autoantlgen complex. Since cellular modulatmn of Ro/SS-A anngen expressiOn has been implicated I” the pathogenesls of subacute cutaneous lupus ervthematosus ILE) and neonatal LE. we have begun to examine the genetuz regulation of CR. To examine transcriptlonel regulation of CR. a reporter gene construct was developed bv msertino a 51 1 bo fraoment of the 5’ flsnkino reoion of a oenomic clone of CR & a- &&d co&i& a bacterial chloramphenicol ace~vltransferase (CAT1 reoorter wane (CR-CAT). CR-CAT was then transfected Into A431 cells and relect,ve-&wth ,n G418 was used to isolate three clones that were confIrmed bv polymerase cham reactionandSouthernblatting tohave CR-CA7sfablyintegrated intooenomlc DNA. Each of the three cloneswa~ to constitutively express
found
low
OF
avels
of CAT
activitv
as detected
bv thin
Iaver chromatowaohv.
Calcium
&ophore shock lAZ3lSi 2.5 @firnIl. heat shock l43OC x 1-W and Zinc ion exoosure (225 uMI were found to mcrease CAT expression in these clones bV SOOl.iOO%. 400.‘600% and 500.1.700% respect,ve,V. Studies are underwav to exam,ne the effects of “ltrav,olet B hght on CR-CAT express,on These reSults suggest that al the 51 fragment of the 5’ regulatory reg#on examined in this study 1s sufflclent for promoting transcrlptlon of the human CR gene, bl CR is const~tuwelv transcribed I” a transformed epidermal kerat8nocyte hne, c) CR is regulated at the transcrlptlonal level, and d) CR, like several other LE-related autoant,gens, appears to function as a heat shock gene
1bp
C’IVDYCF ZTEilOIL SULii,WBE GWE LiELETIONS ANI: CEATECTION OF THE WRRIWS &ilan Hu and Li Fang, Departiaent of Lernatology. tiua?%an iiospital, Shanghai Nedice. University, Shanghai, F.R.ihina Recessive X-linked Ichthyosis(RXI.1) is one of the nest common x-linked genetic disorders, which is attributed to deficiency of steroid sulfatase(STS). In approximatelv W% of the natients. this lack is due to the deletion of 53 gene-which is located at Xp22.3. The &.A probe of STS gene has been isolated previously by other cmups. For the first time in China, we analyzed 19 ca*e* of RALI ~IYJ 11 cases of Autosomal Lnminant IchthyosisjiLI) oy Southern olot hybridization and Polymense 3hain ReactloniPCR) as well. We found that 84% (16 out of 19) FXLI have STS gene deletions vhile kL1 haven't any. iurtnermore, as the carriers have half-normal gene dosage, we have used gene dosage analysis to detect the carriers in four RXLI families and found two "others be hetemzygote. These experiments contributed to rapid. simple and effective gene diagnosis and prenatal diagnosis of R.LI.
434