Mitotlic Instability of Transformants
David H. Griffin
griffin at rodan.syr.edu
Fri Dec 3 15:00:26 EST 1993
I received several worthwhile responses to my inquiry on mitotic
instability of transformants. I would like to thank all that replied.
Below is a summary of the information that I obtained from these responses
and some additional references I found as a result of this information.
It seems that there is quite a variety of responses by individual fungi
and several different levels of instability, indicating interesting
variation in the mechanisms controlling recombination during mitotic growth.
Keller N P, G C Bergstrom and O C Yoder. 1991. Mitotic stability of
transforming DNA is deter;mined by its chromosomal configuration in
the fungus Cochliobolus heterostrophus. Curr Genet 19:227-233.
Cochliobolus heterostrophus was transformed with pH1S confering
resistance to hyg B under the control of an 838 bp promoter fragment
from C. heterostrophus. Plasmid integration was either at homologous
sites (52% single copy, 33% tandemly repeated copies) or ectopic sites
(4% single copy, 11% tandem repeats) resulting in four distinct
configurations of integrated DNA. All four were stable during mitotic
growth with no loss of integrated DNA after five subcultures on
nonselective media or seven cycles of pathogenesis on maize. However,
after eight or more passages on maize deletion of integrated DNA was
detected with the frequency of deletion depending on the configuration of
the integrated DNA. A single copy flanked by direct repeats of target,
homologous-site sequences was least stable and a single copy at an
ectopic site with no flanking repeats was most stable. Tandemly repeated
copies at either homologous or ectopic sites had intermediate stabilities.
Cytosine methylation occurred during mitotic growth, but had no effect
on expression of hygB.
Arnau J and R P Oliver. 1993. Inheritance and alteration of
transforming DNA during an induced parasexual cycle in the imperfect
fungus Cladosporium fulvum. Curr Genet 23:508-511.
Protoplasts of a pAN7-1-transformed isolate of Cladosporium fulvum
race 4, harboring a tandem duplication of the vector, and an
untransformed race 5 isolate were fused, without selection for the
presence of vector sequences. Fusion products were allowed to haploidize
spontaneously. The inheritance of pAN7-1 sequences and the expression
of the hph gene was studied in 85 progeny. A very high proportion
(80%) of the progeny contained vector sequences and 70% of the
progeny were resistant to hygromycin. Inactivation of the hph gene
occurred in eight progeny. Rearrangement of vector sequences can
account for the inactivation. There is no evidence for any RIP-like
mechanism. Several novel bands were observed.
Rikkerink, E.H.A., S.L. Solon, R. . Crowhurst, and M.D. Templeton.
1993. Integration of vectors by homologous recombination in the plant
pathogen Glomerella cingulata. Current Genetics, In Press.
In this paper we describe how transformants of G. cingulata, derived by
homologous recombination of a circular vector with homologous gpdA
sequences, appear to be quite unstable. Approximately 10-20% of the
conidia derived from such transformants lose their hygromycin
resistance by recombination between the (now repeated) homologous
sequences, and wild type fragments are clearly visible in Southerns.
These figures do not take into account any fluctuations due to founder
effect, so they can't be taken as a true indication of the frequency but it
is obviously high. Interestingly there may be some influence by the
length of the repeated DNA since decreasing the length of repeated DNA
from 1.5 kb to 0.5 kb resulted in a marked decrease in the frequency of
this deletion event to the point where the deletion event is no longer
detectable by Southerns. It should be kept in mind that we found a very
high rate of homologous recombination at the gpdA locus in G. cingulata
(95% of our transformants were homologous integrants) and that the
unusually high frequency of homologous recombination in G.cingulata
could be responsible for this effect.
We haven't done any detailed studies of mitotic stability of multiple
Klaas Swart (Dept. Genetics, Agricultural University, Wageningen) and
coworkers studied mitotic stability of single and multiple integrants in
Aspergillus niger transformants. Some are mentioned in: Debets, Swart,
Holub, Goosen and Bos, 1990 Genetic analysis of amdS transformants of
A.niger and their use in chromosome mapping. Mol Gen Genet 222: 284
- 290. Although he not worked on this recently, this is still a subject of
interest in his lab.
Additional references located referring to instability phenomena with
Berges T and C Barreau. 1991. Isolation of uridine auxotrophs from
Trichoderma reesei and efficient transformation with the cloned ura3
and ura5 genes. Curr Genet 19:359-365.
Blakemore E J A, M J Dobson, M J Hocart, J A Lucas and J F Peberdy.
1989. Transformation of Pseudocercosporella herpotrichoides using
two heterologous genes. Curr Genet 16:177-180.
Bussink H J D, J P T W van den Hombergh, P R L A van den IJssel and J
Visser. 1992. Characterization of polygalacturonase-overproducing
Aspergillus niger transformants. Appl Microbiol Biotechnol 37:324-
Faugeron G, L Rhounim and J L Rossignol. 1990. How does the cell count
the number of ectopic copies of a gene in the premeiotic inactivation
process acting in Ascobolus immersus? Genetics 124:585-591.
Feher Z, M Schablik, A Kiss, A Zsindely and G Szabo. 1986.
Characterization of inl+ transformants of Neurospora crassa obtained
with a recombinant cosmid-pool. Curr Genet 11:131-137.
Kistler H C and U K Benny. 1988. Genetic transformation of the fungal
plant wilt pathogen, Fusarium oxysporum. Curr Genet 13:145-150.
Pukkila P J and C Skrzynia. 1993. Frequent changes in the number of
reiterated ribosomal RNA genes throughout the life cycle of the
basidiomycete Coprinus cinereus. Genetics 133:203-211.
Ruiz-Sala P, J A Perez-Gonzalez and D Ramon. 1993. Nucleotide
sequence of a Trichoderma longibrachiatum DNA fragment encoding the
5.8S rRNA gene. Nucl Acids Res 21:741.
Smith T L, J Gaskell, R M Berka, M Yang, D J Henner and D Cullen.
1990. The promoter of the glucoamylase-encoding gene of Aspergillus
niger functions in Ustilago maydis. Gene 88:259-262.
Unkles S E, E I Campbell, D Carrez, C Grieve, R Contreras, W Fiers, C A
M J J Van den Hondel and J R Kinghorn. 1989. Transformation of
Aspergillus niger with the homologous nitrate reductase gene. Gene
Upshall A. 1986. Genetic and molecular characterization of argB+
transformants of Aspergillus nidulans. Curr Genet 10:593-599.
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