Prokaryote genes do not include introns, while most eukaryote genes do. It is for
this reason that brown algae, which are relatively easy to manipulate, have chiefly
been used to date.
Many brown algae possess plasmids (extra-chromosomal genes capable of
autoreproduction). Because of uncertainties concerning their physiological functions, however, a marker must be introduced for use as a vector (DNA used to
transport foreign DNA into a host).
For example, the R2 strain of Anacystis nidulans, which possesses the integral
plasmid pUH24 (7.8 kbp), has been transformed with the E. coli. plasmid pRI 46,
which contains Tn901, a transposon (a sequence that moves chromosomal genes or
plasmids) coding for ampicillin resistance (Ap
r ). While the frequency was very low,
manipulated pUH 24::Tn 901 plasmids (that is, where Tn 901 has been shifted to
pUH24) have been found to occur in analysis of five Ap
r transformation strains.
Because Tn 901 shifting is an issue in the use of this recombinant plasmid as a
vector, it was truncated with the restriction enzyme BamHI and the transposon
fixed. The resulting plasmid (called pUC1) possessed one section each of BamHI
and XhoI and two of Bgl II and coded for Ap
r
. Cloning of several blue-green algae
vectors in this way allowed the production of mutant strains with drug-resistant
properties.
One example involved the use of the A. nidulans R2’s pUH24 and the colon
bacillus vector pACYC to produce a shuttle vector (a vector capable of independent
growth on cells from either of two organisms) and pUC 303 (tolerance to Cm
r , Sm
r ,
chloramphenicol and streptomycin), which were combined with an A. nidulans R2 Á
pUH24 delation strain. The result was a high-efficiency transformation in which
drug tolerance remained stable for a long period of time.
Cloning of acetate reductase in A. nidulans R2 has also been achieved with the
comid vector pPUC29, which includes a phage DNA cos region in the shuttle
vector.
Because cosmids have properties intermediate between those of phage vectors
and plasmid vectors, DNA fragments of up to 50 kbp can be introduced and a gene
library (including recombinants of all chromosomal DNA factors with vectors and
all chromosome pair regions) can be produced.
The above is an example of genetic manipulation through transformation. Other
forms of manipulation have attempted through electroporation, or the use of an
electric pulse to produce a tiny hole in the cell membrane through which genes can
be physically introduced, and, in the case of filamentous blue-green algae such as
Anabaena sp. or Nostoc sp., through conjugative transfer using colon bacilli.
DNA introduced in transformation may be used under fully in vitro conditions.
When a restriction enzyme line exists in the host, transformation may be achieved
by altering the DNA beforehand with the corresponding methylase. In the conjugative transfer method, however, the DNA introduced is transformed and cloned
within the E. coli, and lines transformed in vitro cannot be used.
One approach used to address this issue has been to clone methyl transferase
DNA as a helper plasmid for conjugation. Transformation is performed in vivo and
conjugated with the blue-green alga. This method has been used for conjugative
212
7 Microalgae, a Biological Resource for the Future
this reason that brown algae, which are relatively easy to manipulate, have chiefly
been used to date.
Many brown algae possess plasmids (extra-chromosomal genes capable of
autoreproduction). Because of uncertainties concerning their physiological functions, however, a marker must be introduced for use as a vector (DNA used to
transport foreign DNA into a host).
For example, the R2 strain of Anacystis nidulans, which possesses the integral
plasmid pUH24 (7.8 kbp), has been transformed with the E. coli. plasmid pRI 46,
which contains Tn901, a transposon (a sequence that moves chromosomal genes or
plasmids) coding for ampicillin resistance (Ap
r ). While the frequency was very low,
manipulated pUH 24::Tn 901 plasmids (that is, where Tn 901 has been shifted to
pUH24) have been found to occur in analysis of five Ap
r transformation strains.
Because Tn 901 shifting is an issue in the use of this recombinant plasmid as a
vector, it was truncated with the restriction enzyme BamHI and the transposon
fixed. The resulting plasmid (called pUC1) possessed one section each of BamHI
and XhoI and two of Bgl II and coded for Ap
r
. Cloning of several blue-green algae
vectors in this way allowed the production of mutant strains with drug-resistant
properties.
One example involved the use of the A. nidulans R2’s pUH24 and the colon
bacillus vector pACYC to produce a shuttle vector (a vector capable of independent
growth on cells from either of two organisms) and pUC 303 (tolerance to Cm
r , Sm
r ,
chloramphenicol and streptomycin), which were combined with an A. nidulans R2 Á
pUH24 delation strain. The result was a high-efficiency transformation in which
drug tolerance remained stable for a long period of time.
Cloning of acetate reductase in A. nidulans R2 has also been achieved with the
comid vector pPUC29, which includes a phage DNA cos region in the shuttle
vector.
Because cosmids have properties intermediate between those of phage vectors
and plasmid vectors, DNA fragments of up to 50 kbp can be introduced and a gene
library (including recombinants of all chromosomal DNA factors with vectors and
all chromosome pair regions) can be produced.
The above is an example of genetic manipulation through transformation. Other
forms of manipulation have attempted through electroporation, or the use of an
electric pulse to produce a tiny hole in the cell membrane through which genes can
be physically introduced, and, in the case of filamentous blue-green algae such as
Anabaena sp. or Nostoc sp., through conjugative transfer using colon bacilli.
DNA introduced in transformation may be used under fully in vitro conditions.
When a restriction enzyme line exists in the host, transformation may be achieved
by altering the DNA beforehand with the corresponding methylase. In the conjugative transfer method, however, the DNA introduced is transformed and cloned
within the E. coli, and lines transformed in vitro cannot be used.
One approach used to address this issue has been to clone methyl transferase
DNA as a helper plasmid for conjugation. Transformation is performed in vivo and
conjugated with the blue-green alga. This method has been used for conjugative
212
7 Microalgae, a Biological Resource for the Future