الفهرس 
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Abstract
SUMMARY
The main obj ecti ve of this envestigation was to
construct stable highly efficient 0( -amylase producer
saccharomyces cerevisiae strains through the acquiring
of genes responsible for this enzyme production.
Two haploid yeast strains of saccharomyces
cerevisiae and one diploid wild type strain were used
as recipients in this investigation.
Six organisms donor strains were used as a source
of DNA (t>< -amylase production); Bacillus subtilis
(CAlM 1007), Bacillus thuriengiensis (1), Bacillus
T huriengensis (2), Bacillus sp , , Aspergillus oryzae
and Aspergillus niger.
This study was conducted during 1991, 1992 and
. 1993 in the Genetic lab. Faculty of Agriculture,
Moshtohor, Zagazig University.
The results of this study were divided into four
parts and summarized as follows:-
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SUMMARY
1- Selection of highly amylase-produces donors:
Bacillus subtilis (CAlM 1007) strain was the highly
eff f.caent strain for producing c< -amylase enzyme. This
strain had the large of zone diameter (em) for starch
utilization comparing with the other donor strains,
therefore the curde lysate DNA of these strains used
in the transformation experiments.
11- Genetic transformation:
Three methods of transformation were used with
DNA which isolated by two methods. (1) No transformants
were obtained by using DNA which isolated by two methods
without adding any alkali cations on the selective media.
The failure of transformation in this respect may
be due to absence of alkali cations which
removed the thick cell wall of yeast. (2) Using DNA
+ 0.3% lethium acetate as alkali cation, the haploid
strain A 1 (~ cerevisiae) exhibited a transfol”’llecCellls
with crude lysate DNA of Bacillus subtilis. Traaforution
frequency was 2.-6xlO-5S- The other recipient strains
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SUMMARY
B1 and diploid not exhibited any transformants. It may
be due to the ability of the genetic constitution of
the haploid strain Al to transformed in the presence
of lethium acetate.
(3) Using DNA + 24% polyethylene glycol (PEG) +
0.3% lethium acetoite + Tris-Hcl + EDTA, the haploid
strain Bl of yeast exhibited a transformants with crude
lysate DNA of ~ subtilis. The transformation frequency
was 0.00015%. With diploid strain the transformation
frequency was 0.0001%.
The haploid strain Al exhibited transformation
frequency higher than the other haploid strain Band
the transformation frequency of the haploid strain
Al or B1 were higher than the diploid strain.
The DNA was mechanically fragmented using
microsyringe failed to exhibited any transfo~ts
with O.3X lethium acetate. This 118ybe due to the loss
of gene (s) responsible for allJ’lase production as a
result of DNA repurifications.
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SUMMARY
III-Determination of Do<; -amylase activity:
Two methods were used to determine :::<-amylase
activity. (Qua!ttatively and Quantitatively)
(l) The qualitative method was determined by using
the vapour of iodine and showed that mean of zone
diameter of S. cerevisiae transformed were higher in
the haploid strain (Al ) transformants tr3. Tr2,Trl
and Tr4t respectively than the haploid strain (B 1 )
transformants and in the diploid transformants.
(2) The quantitative method was determined
calorimetrically. All Bl transformants were higher than
the A
l
transformants in reducing sugar. The haploid
transformants A1 and Bl were higher than the diploid
yeast in the reducing sugar.
IV- Plasm d curing:
Gene (s) responsible for the stablity of transformation
may be located in pl.asm.d (extra-chra-osOll81
DNA).
No clear zone after exposure to iodiDe vapour with
SDS (SodiWll Dodecyl sulfate) indicates that pIa.-id
which carried genes responsible for _ylase production
was cured by SOO.
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