Chloroplasr DNA and Drug Resistance

When Ruth Sager place Chlamidomonas algal cells on a culture medium

containing thw antibiotic strepyomycin, most of the cells were killed, but about
one per million survive and multiplied, each to form a streptomycin- resistant
colony. Mutatnt with resistance to streptomycin were being selected from the
predominanly streptomycin- susceptible algae. About 90 pecent of mutants
involved nuclear genes (sr-1), and such mutation were merely being
demonstrated by the antibiotic challenge. Approximately 10 percent ofthe
mutation (sr-2), however were uniparental and nonchromosomal. Eventually,
nonchromosomal mutan were recovered from almost every colony. Non
chromosomal DNA mutations expressed the same phenotypes as chromosomal
DNA mutants. These non chromosomal genes are presumed to be located in the
chloroplasts.
Reciprocal crosses (fig. 20.5) demonstrated that antibiotic resistance,controlled
by nonchromosomal gene,was uniparental in inheritance. On the other hand,
mating type in this sexual unicellular alga was controlled by chromosomal genes,
ehich were designated by the investigators mt + and mt- or simply plus (+) and
minus (-). Instead of female and male. All progeny from each reciprocal mating
were like the plus (+) mating type with respect to relative streptomycin
resistance thus demonstrating maternal inheritance. When the plus (+; female)
matingtype was resistant, all progeny were resistant : when plus (+)mating type
was nonresistant, all progeny were nonresistant. These result ofreciprocal
crosses demonstrated non- Mendlian inheritance, which involved a single pair of
contrasting traits. Nonchromosomal genes, sr for streptomycin resistance and ss
for streptomycin sensitive, were postulated to control these two alternative
characteristic.
Another mutant, ac2, which blocked photosynthetic, was induced and a pair of
nonchromosomal alleles, ac1, and ac2, was thus available for study in same strain
of Chlamydomonas. The mutant required acetate in the medium for growht. With
two pairs ofof non chromosomal genes available, a dihybrid type ac 1 ss x ac2 sr
were prepared, and progeny were allowed to grow for a few vegetative
multiplications. Each cell was thesn classified for itssegregating makers, both
nonchromosomal and chromosomal(i.e, ers, mating type and others known to be
chromosomal). Both the ac1/ac2and the sr/sspairs off alleles were observed to
segregate, but not always in the same division. After four of five mitoti doubling,
both parentals (ac1 ss and ac2 sr) and recombinants (ac1 ss and ac2 sr) had been
obtained. The result indicated independent assortment, suggesting that two
pairs of nonchromosomal genes werecarried in different plastids. Three and four
point crosses and reciprocal crosses have been made withthe addition of several
mutants, which are presumedto be carried in chloroplasts and mitochondria. A
genetic map of non Mendeliangenes in Chlamydomonas has been constructed,
but uncertainty still exist as to whether some chloroplast linkage groups are
solely in the clhoroplast genome.
Organization of Plastid Genomes

The plastid genomes of over 200 species of higher plants and of many bluegreen, and red algae have been a least partially characterized. Within a given
species, the genomes of different types of plastids- chloroplast, amyloplast
(plastid that accumulate starch in storage tissues), and chromoplasts (plastid
containing pigments)- allare identical in organisms where they have been
studied. Thus our discussion of plastid genome structure will be restricted to the
organization of the DNAs of chloroplast (cpDNAs)- the most important member of
the plastid family.
In higher plants, cpDNAs range in size range for chloroplast genomes is much
larger from 85 to 292kb for species known to have circular cpDNAs. In two
species of green algaes of the genus Acetabularia, the cpDNAs apper to be huge,
about 2000 kb, and it has not yet been established whether these large
chloroplast genomes are linear or circular. As in the case of mitochondrial DNAs,
chloroplast often contain multiple copies of the cpDNA. The single- celled
flagellate Euglena gracilis contains about 15 chloroplast each with about 40
copies of cpDNA, giving a total of about 600 copies per organism.
All the chloroplast genomes analyzed to date contain basically the same set of
genes, but with these genes arranged in very different ways in th cpDNAs.the
genes present on cpDNAs can be grouped into two major classes : (1)bthosethat
encode components ofthe chloroplast protein biosynthetic apparatus(RNA
polymerase subunits, stuctural components of chloroplast ribosomes, and a set
of tRNAs) and (2) those specifying components of the photosynthetic machinery
(photosystem I and II and the electron transprt chains).
Chloroplast genomes of higher plants are about one twentieth to one thirtieth the
size of the genomes of the prokaryotic organism (blue-green algae or
cyanobacteria) from which they are believed to have envolved. Thus, the
clhoroplast hve lost much of the genetic information of their ancestors and have
become very dependent on nuclear genes of the host cell for many essential
components. As in the case of mitochondria, the latter components are
synthesized on cytoplasmic ribosomes and are synthesized on cytoplasmic
ribosomes and are imported into chloroplast wiht the aid of amino-terminal
transit peptides that are cleaved off during transport through the chloroplast
membranes.
Comparative studies of chloroplast genomes have provide important new
information abour evolutionanary realtionships of plant and alga species J.D
Palmer has distinguished six major lines of chloroplast evolution. The chloroplast
genomes present in different evolutionary lines all contain largely the same
genes, but these genes are present in different arrangement o the cpDNA
molecules. The rRNA genes are present in duplicate on inverse repeats repeats
within the cpDNAs of most species. However, in Euglena gracilis, the 16S and
23S rRNA genes are present on three direct tandem repeats, wit a separate
fourth copy of the 16S rRNA gene located nearby in the genome. By comparing
the locations of genes are present on three direct tandem repeats, with a
separate fourth copy of the 16S rRNA gene located nearby in the genome. By

comparing the locations of genes on different chloroplast genomesm Palmer

hasbeen able to show that many of the changes in cpDNA organitation have
resulted from inversions of segments of DNA were found to have occured in
intergenic regions ang within introns of genes. However, the major documented
changes in cpDNA structure seem to have resulted from large inversions.
Photosynthesis occuring within chloroplast provide the life sustaining energi
source for all living organisms on planeth Earth. Since chloroplast genomenes
encode many key component of photosystem I ang II and the electron transport
chains, knowledge of the structure and fuction of cpDNAs is very important and
has receive much attentio. The complete nucleotide-pair sequence of the
cpDNAs of Marchantia polymorpha and tobaco (nicotiana tobacum) have been
determined. The cpDNAs of Marchantia polymorpha and tobaco are 121,024 and
155,844 nucleotide- pair in lenght, respectively. The organization of the singlecopy genes in the chloroplast genomes of these two plants is remarkably similar
considering that they are evolutionary very distant from each other. The major
different between these two cpDNAs is that the inverse repeat regions containing
the rRNA genes are considerably larger in tobacco. The best estimate of cpDNA
gene number are 136 in Marchantia and 150 in tobacco. The locations of known
genes and open reading frames are shown for the chloroplast genome of
Marchantia
Perhaps a complete understanding of the chloroplast genes and the product that
they encode will have important pratical applications in the future. Information of
the exact mechanisms by which photosystem I and II function might someday
permit scientists and engineers to build a totally synthetic system capable of
duplicating the capacity o green plants to capture light energy and convert it to
chemical forms useful to living organisms.