Although the eDNA sequences for the 10 k:Da (hsp 10, hsp 1 0) and the 60 k:Da (hsp60,
cpn60) heat shock proteins have been obtained for a number of mammalian species, until very
recently information was not available on the functional genes encoding these proteins. The
primary objective of this work was to clone and sequence the functional genes for these
proteins from CHO, Chinese hamster ovary cells. Screening of a lambda EMBL3 CHO
genomic library with the CHO hsp 10 eDNA identified a clone containing the putative hsp 10
functional gene. A -5.5 kb fragment was isolated from one of these clones by enzymatic
digestion and -3.3 kb was sequenced. The clone was found to contain consensus regulatory
sequences upstream of the putative transcription initiation site, + 1, including two Sp 1 binding
sites, a CAAT box, and a single heat shock element, HSE, but lacked a TATA box. The
coding region consists of four exons, identical to the hsp10 CHO eDNA sequence, separated
by three introns, of 200 bp, 600 bp and 1600 bp in size, containing conserved splice sites.
Screening of the same EMBL3 CHO genomic library with the CHO hsp 10 eDNA also
resulted in isolation of a full length processed pseudogene with -90 % identity to the eDNA.
This pseudogene lacked introns, contained a poly(A) tract, as well as various single bp
changes, additions and deletions. The upstream region of this pseudo gene was found to
contain similarity to the human LINE sequence, a DNA repetitive element. PCR amplification
ofCHO-WT genomic DNA resulted in isolation offive additional processed pseudogenes,
corresponding to the central -270 bp of the CHO hsplO eDNA. All the pseudogenes
displayed a high degree of similarity to the CHO hsp 10 eDNA sequence despite the presence
of numerous mutations. Prior to this report, pseudogenes had not been found associated with
hsp 10. The identification of these pseudogenes suggests the presence of a multi gene family
for this heat shock protein in the CHO genome.
Previously, a semi-processed pseudogene, Gel, was identified for hsp60 from CHO
cells which contained a single -87 bp intron near its 3' end (Venner eta/., 1990). From this
pseudo gene, a fragment containing the -87 bp intron was isolated for use as a probe to screen
a lambda EMBL3 CHO genomic library. This resulted in isolation of several positive clones,
two of which were purified, a -1.0 kb fragment amplified by PCR and then sequenced
revealing two additional semi-processed pseudogenes, designated .A4 and .AS. These
pseudo genes were found to be homologous to the GC 1 clone, containing many similar
mutations as well as the -87 bp intron. Utilizing CHO-WT genomic DNA, a separate PCR
amplification resulted in isolation of a -2.5 kb fragment which was partially sequenced and
found to correspond to the putative hsp60 functional gene. The fragment contained one
exon, which was identical to the CHO hsp60 eDNA in the region sequenced, and two introns
of800 bp and 1500 bp. This fragment can now provide an ideal probe for isolation ofthe
CHO hsp60 functional gene. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22676 |
| Date | 12 1900 |
| Creators | Zurawinski, Joni |
| Contributors | Gupta, R.S, Biochemistry |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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