Fungal cells with inactivated DNA mismatch repair system
A technology of mismatch repair and DNA sequencing, applied in DNA preparation, recombinant DNA technology, fungi, etc., can solve the problem of library diversity limitation
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Embodiment 1
[0131] Gels suitable for determining whether filamentous fungal cells are inactivated in a mismatch repair system as described herein:
[0132] The principle of this gel shift (shift) assay is that cell extracts are made from: (a) filamentous fungal cells in which genes involved in the mismatch repair system as described herein are inactivated; and (b) Corresponding filamentous fungal cells in which the genes are not inactivated.
[0133] These extracts were then combined / mixed with base pair mismatched G:T, G:A, G:G, A:C oligonucleotides and supercoiled TG dinucleotides in a non-denaturing gel. measured on the glue.
[0134] If the gel shift (shift) assay demonstrates that the control filamentous fungal cells containing genes that are not inactivated contain any protein that binds to any of the above oligonucleotides, and these binding proteins participate in the mismatch repair system as described herein If it is not seen in the filamentous fungal cells whose genes have be...
Embodiment 2
[0148] Cloning of genes involved in the mismatch repair system of Aspergillus oryzae cells
[0149] The gene cloned as described in this example is shown in SEQ ID NO 1 (DNA sequence) and SEQ ID NO 2 (translated amino acid sequence).
[0150] Several sequences of mismatch repair proteins from different organisms are known, only three of which are used below: Saccharomyces cerevisiae (M84170), H. sapiens (L47580) and mouse (U21011).
[0151] The numbers shown are queries of the publicly available GenBank database.
[0152] Based on the C-terminal homology between known mismatch repair proteins, a set of simple heterogeneous primers was designed to amplify the incomplete sequence of the Aspergillus oryzae homologue:
[0153] Pr117858 (SEQ ID NO 10): P-GGCNCARATHGGNTGYTTYGTNCC
[0154] Pr117859 (SEQ ID NO 11): P-GCCCANGCNARNCCRAANCC
[0155] Using chromosomal DNA from Aspergillus oryzae strain JaL142 (WO96 / 29391) as a template and the above primers, eight MgSO 4 The following...
Embodiment 3
[0178] Destroy the cloned gene of Example 1 on the Aspergillus oryzae cell chromosome:
[0179] For disruption experiments, the msh2 CDS was deleted by PCR from pUC19msh2P (see Example 2) and a NotI site was introduced instead. Primers for this reaction are:
[0180] 137208 (SEQ ID NO 24): 5'P-CCGCGTCTCCAACAAGATGAATGG
[0181] 137207 (SEQ ID NO 25): 5'P-CCGCTTTCTCGGGGTCATAGC
[0182] With 2.5mM MgSO 4 and 50 pg pUC19msh2P in a Pwo polymerase-based PCR reaction (according to the manufacturer's recommended conditions):
[0183] The PCR cycle is: [96°C 2min-(94°C 30s; 52°C 30s; 72°C 3min) cycle 4 times-(94°C 30s; 59°C 30s; 72°C 3min) cycle 25 times-72°C 10min].
[0184] The resulting PCR product of about 8.9 Kb was analyzed, ligated into pUC19, and then transformed into Escherichia coli XL1. pMsh2Δ was analyzed from the resulting transformants and the correctness of the new junction and its surrounding points were verified by sequencing [Primer 138149 (SEQ ID NO 26): CCTTTCC...
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