Alpha complementation of LacZ in mammalian cells
Peter
Moosmann
and
Sandro
Rusconi
1,
*
Institut für Molekularbiologie II, Winterthurerstrasse 190, CH-8057
Zürich
,
Switzerland
and
1
Institut de Biochimie, Université Fribourg, Pérolles, CH-1700
Fribourg
,
Switzerland
Received January 9, 1996;
Accepted February 7, 1996
The bacterial lactose converting enzyme beta-galactosidase ([beta]-gal) and its gene (LacZ) have been studied for many years (
1
and references therein) and are among the most utilized tools in molecular
biology. The LacZ product, a polypeptide of 1029 amino acids, gives rise to the
functional enzyme after tetramerization (
2
and references therein) and is easily detected by chromogenic substrates either
in cell lysates or directly on fixed cells
in situ
(
3
and references therein). The tetramerization is dependent on the presence of
the N-terminal region spanning the first 50 residues (
2
and references therein). Deletions in the N-terminal sequence generate a so-called omega peptide that is unable to tetramerize and does not
display enzymatic activity. The activity of the omega peptide can be fully
restored either in bacteria or
in vitro
(
4
) if a small fragment (called alpha peptide) corresponding to the intact N-terminal portion of the [beta]-gal is added
in trans
. The phenomenon is called alpha complementation and the small N-terminal peptide is called alpha peptide. This effect has been widely
exploited for studies in procaryotes, where special strains that constitutively
express omega peptide exist and allow the detection of expression of the small
alpha peptide.
Aiming to study the stability of microsatellites in ageing eucaryotic cells, we
have engineered (CA)
n
repeats at the N-terminal position of LacZ (Fig.
1
A). However, we were disappointed by the rapid loss of activity caused by
prolonged repeats (see below). We reasoned that, analogously to procaryotic
systems, the alpha peptide might be more stable towards such insertions.
Surprisingly we could not find any report describing alpha complementation of
LacZ in mammalian cells. Therefore, we constructed eucaryotic expression
vectors that should produce either a lacZ omega peptide [Fig.
1
A, construct g, called Z(d)NC] or different alpha peptides (constructs b-d, called Z-N58, Z-N85 and Z-N150, respectively). We have also compared the
tolerance towards N-terminal inserts for the whole enzyme [construct f, Z(1)NC] or for an
alpha peptide [construct e, Z(i)N58]. Transfection of these constructs in
various combinations has produced the results shown in Figure
1
B, where the length of the bars represents the relative enzymatic activity
obtained from extracts of transiently expressing HeLa cells. The data clearly
show that co-expression of alpha and omega peptides (lines 4-6 and 8-12) resuscitates enzymatic activity that is absent in the
single components (see control lines 2 and 3). We find that the optimum length
of the alpha complementing peptide is ~85 amino acids (line 5), while a shorter peptide (truncated at position 58)
that is otherwise active in most prokaryotic systems (see Fig.
1
legend) demonstrates only a very weak alpha complementing activity (line 4). We
could also demonstrate that alpha peptides are more tolerant towards insertions
of foreign sequences (compare lines 8-12 with lines 13-15). The tolerance was tested with the relatively weak N58 alpha
complementing peptide and we expect the N85 construct to be even more tolerant. We are looking forward to using similar
peptides to monitor stability of dinucleotide repeats in mammalian cells.
Recently we learned that expression of Z-N85 and Z(d)NC in yeast cells does also result in efficient
trans
complementation (D. Picard, personal communication). We observed the same complementation behaviour in other human cell lines (S. B.
Verca, unpublished). Therefore, we are confident that these constructs can be
used in a variety of eucaryotic cells.
REFERENCES
1 Ullmann, A., Jacob, F. and Monod, J. (1967) J. Mol. Biol., 24, 339-343.MEDLINE Abstract
2 Jacobson, R. H., Zhang, X.-J., DuBose, R. F. and Matthews, B. W. (1994) Nature, 369, 761-766.MEDLINE Abstract
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4 Gallagher, C. N., Roth, N. J. and Huber, R. E. (1994) Prep. Biochem., 24, 297-304.MEDLINE Abstract
5 Rusconi, S., Severne, Y., Georgiev, O., Galli, I. and Wieland, S. (1990) Gene, 89, 211-221.MEDLINE Abstract
