Subject: Mitotlic Instability of Transformants

From: [email-masked] (David H. Griffin)
Date: Fri, 3 Dec 93 15:00:26 EST

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 
integration events.

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 
integrated sequences:

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-
329.

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 
78:157-166.

Upshall A. 1986. Genetic and molecular characterization of argB+ 
transformants of Aspergillus nidulans. Curr Genet 10:593-599.