NR APTE
AU Doh-ura,K.; Perryman,S.; Race,R.E.; Chesebro,B.
TI Differently expressed genes in scrapie-infected neuroblastoma cells
QU Internet-Poster DE0405
IA http://www.medic.mie-u.ac.jp/posters/DE0405/DE0405int.html
PT Poster
VT
Summary
To identify genes with altered expression associated with scrapie infection in vitro cDNA libraries from scrapie-infected and non-infected murine neuroblastoma cell lines were screened with cDNA probes derived by subtractive hybridization from scrapie-infected and uninfected cells. Eleven independent recombinant clones whose expression was either increased or decreased in scrapie-infected cells were identified. Expression of these genes was analyzed in a panel of other scrapie-infected and non-infected cell lines. Five genes had altered mRNA exprssion in most scrapie-infected neuroblastoma cell clones compared to non-infected clones. These contained four identified genes and one unidentified gene.
KW scrapie, neuroblastoma cell, differential screening, Northern blot
Introduction - "/posters/DE0405/DE0405int.html"
Scrapie is a transmissible spongiform encephalopathy of sheep and goats characterized by a long incubation period followed by a progressive illness involving degeneration of the central nervous system. Although a lot of research has been done on the pathogenesis of the disease, the nature of the transmissible agent remains obscure. In order to look for new genes encoded by a possible exogenous agent or host genes whose expression might be altered by scrapie infection, cDNA clones whose expression was modified in scrapie-infected murine neuroblastama cells were isolated by comparing mRNA species in scrapie-infected and uninfected cells using differential screening of libraries with absorbed cDNA probes.
Materials and Methods "/posters/DE0405/DE0405met.html"
Cells. Scrapie-infected (J5, 325, J5I, J54, KBE) and uninfected (h-CK, CK, MF, 508, 509, CCL) mouse neuroblastoma cell lines were grown in minimal essential media supplemented with 10% fetal bovine serum. cDNA library construction. Double-stranded cDNA was synthesized from J5 mRNA and h-CK mRNA using oligo(dT) and random hexamer primers. The cDNA was ligated into Lambda ZAPII vector and packaged in vitro. Absorbed hybridization probe. Single stranded cDNA from J5 mRNA and h-CK mRNA were absorbed with h-CK mRNA and J5 mRNA respectively, as described by Sive and St. John (Nucleic Acids Res. 1988;16:10937). Screening. The cDNA library was screened with the 32P-labeled absorbed probe. Positive recombinants of lambda ZAPII were isolated, and excised to generate pBluescript plasmids. The recombinant plasmids were screened differentially with 32P-labeled cDNA probes synthesized from J5 and h-CK. Northern blot analyses. One ug of culture cell mRNA per lane was size fractionated. Transfer, hybridization and wash was done as usual protocol. Sequencing. Double-stranded DNA was used for dideoxy chain termination reaction sequencing. Computer search analyses on DNA sequences were performed.
Results - "/posters/DE0405/DE0405res.html"
Screening for mRNA species with altered levels in scrapie-infected J5 cells.
To detect mRNA species that were increased in expression in the J5 clone of scrapie-infected murine neuroblastoma cells a cDNA library from clone J5 was screened with a DNA probe made from J5 mRNA from which the sequences common to non-infected neruoblastoma cells (clone h-CK) were subtacted. 66 independent cDNA sequences with increased expression in the J5 cell line were identified (I-clones). To detect messages which were reduced in J5 cells compared to h-CK cells, a cDNA library from h-CK was screened with a cDNA probe made from h-CK from which the sequences common to J5 mRNA were removed by subtractive hybridization. 32 independent cDNA sequences with decreased expression in the J5 cell line were identified (D-clones).
Northern blot analyses in J5 and h-CK cells and characterization by DNA sequencing.
Using each of the 98 I- or D-clones as a probe, mRNA expression in J5 and h-CK cells was analyzed by Northern transfer. Eight of 66 I-clones and three of 32 D-clones yielded different signal intensities between J5 and h-CK. The blots using clones I-165, I-147 and I-135 showed the most prominent differences. These eleven clones were sequenced and identified by comparison with known databases. The results were shown in table.
Table : cDNA clones with increased (I-clones) or decreased (D-clones) expression in J5 scrapie-infected nneuroblastoma cells.
Genes were identified by comparing the cDNA sequence to GenBank, EMBL and Brookhaven databases. Altered mRNA expression is shown as the ratio of northern blot band intensity estimated by densitometry using poly A mRNA from scrapie-infected J5 cells and uninfected h-CK cells
Northern blot analyses in other scrapie-infected and uninfected cells.
Both the J5 cell line and the h-CK were passaged in vitro for several years after establishment of scrapie infection in vitro. Due to selective pressure during frequent in vitro passages these cell lines might differ in some properties unrelated to scrapie infection. To address this issue, mRNA from 5 scrapie-infected (Sc+) and 5 uninfected (Sc-) murine neuroblastoma cell lines with different passage histories was analyzed by Northern blotting using the cDNA probes described above. An example of the results was shown in figure. The blots with five cDNA probes (I-165, I-135, I-66, I-126 and I-147) showed increased mRNA expression in most scrapie-infected neuroblastoma cell clones compared to non-infected clones, although the blots with the other cDNA probes showed no significant difference between Sc+ and Sc- cell lines.
Figure : Example of northern blot analyses of Sc+ and Sc- cells.
1ug poly(A)+ RNA from each of the Sc+ neuroblastoma cell lines (MF, CK, CCL, 509 and 508) and Sc+ neuroblastoma cell lines (J5I, J54, 325, KBE and J5) was hybridized with each 32P-labelled cDNA probe. The EF-1 alpha was used as a control for RNA loading and integrity. * indicates residual signals of previous probes which remained after stripping.
Conclusion "/posters/DE0405/DE0405con.html"
In the present experiments, the expression of five genes was significantly increased in most scrapie-infected cell clones compared to uninfected clones, and these results suggested that altered expression of these genes might be caused by scrapie infection of these cells. Our neuroblastoma cell cultures did not contain astrocytes or other brain cell types which are known to show altered gene expression during scrapie infection in vivo. Therefore, the altered gene expression associated with scrapie infection of these cultures probably represents a direct response of neuroblastoma cells to scrapie infection, rather than an indirect effect secondary to astrocyte activation in scrapie-infected brain tissue. Thus, these data may suggest some biochemical clues as to how individual cells react when they are infected with scrapie.
"/posters/DE0405/DE0405com.html"> Comments
Comments on DE0405
Chairman, Dr. : Now, Dicsussion is open.
Send mail directly to office@doc.medic.mie-u.ac.jp instead. Please do not forget including the Poster Number DE0405 in it.
We are looking forward to receiving you message
Date: Fri, 9 Dec 94 18:59:11 JST
E-mail from k-naka@doc.medic.mie-u.ac.jp
Subject: Specific protein synthesis
Dr. Kunio Nakashima says;
Very hard work. I-165 and I-135 expressions seem to be very interesting. Have you tried to analyze the differences in the synthesis of proteins, in relation to those mRNAs?
Date: Fri, 16 Dec 94 23:11:08 JST
E-mail from dohura@doc.medic.mie-u.ac.jp
Subject: comment to Dr Nakashima's question
Dr. Katsumi Doh-ura says;
No, we have not done yet, and we are planning to do that when materials are available.
AD Katsumi Doh-ura, Sylvia Perryman, Richard Race, and Bruce Chesebro in Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, 903 South 4th Street, Hamilton, Montana 59840, U.S.A., Internetadresse: dohura@doc.medic.mie-u.ac.jp
SP englisch
PO Internet
OR Prion-Krankheiten D