Dear Chlamy folks,
Trends in Cell Biology recently invited me to write an article on cloning
genes by 'transformation tagging'. The editors have just returned the
manuscript with the request that more prominence is given to the work of
other labs. and more discussion of the relative merits of ARG7 v. NIT1.
Since only one cloning paper has been published (as far as I'm aware) -
i.e. Tam & Lefebvre (1993), and since all our work has been exclusively
with ARG7, I thought I'd attempt to collect together the experiences of the
various Chlamy labs by sending out this request for information.
Contributions will, of course, be acknowledged and a compilation of the
responses will be posted on the net.
Q.1. Transformation rates:
We typically get 1000-1500 arg+ transformants per plate (i.e using 6x10.7
cells (0.3ml), 0.3g beads, 2ug DNA). How does this compare with NIT1 or
with your ARG7 transformations?
Q.2. Linear v. uncut:
Does linearizing the plasmid improve transformation rates in your hands.
More importantly, do linear plasmids lead to simpler integration patterns
(i.e. does the vector DNA remain intact? - see Q.3).
Q.3. Plasmid (marker) rescue:
Our analysis of several mutants generated using the uncut ARG7 plasmid
reveal that large parts of the pBR329 vector have been lost - hence plasmid
rescue is not possible in these cases. Has anyone succeeded in cloning
flanking sequence by plasmid rescue when using ARG7 (either linear or
uncut)? For those using NIT1, is plasmid rescue the routine method or is
the loss/rearrangement of vector sequence also a problem with this marker.
Q.4. Linkage to the phenotype:
More than half of our mutants don't show co-segregation of the marker and
the mutant phenotype. This is much lower than that reported by Tam &
Lefebvre who scored 13 from 15 mutants generated using NIT1. What sort of
numbers do you get?
Q.5. Tagging by incomplete copies of the marker:
A common occurrence with ARG7 is the integration of incomplete copies of
the marker into the genome in addition to the functional copy. We speculate
that this is the explanation behind our 'untagged' mutants and is an
important consideration when using the arg+ or nit+ phenotype to
demonstrate linkage. Does anyone have any evidence for mutations generated
by non-functional copies of either marker?
Q.6. Anything else:
Does anyone have any other useful tips regarding transformation and the
isolation of tagged genes?
Dr Saul Purton
Dept. of Biology, UCL
E-mail: S.Purton at ucl.ac.uk (Internet)
Phone: 071-387 7050 x2675
FAX: 071-380 7096