Chloroplast DNA and Drug Resistance

When Ruth Sager placed Chlamydomonas alga cells on aculture medium containing
the anibiotic streptomycin, most of the cells were killed, but about one permillion survived and
multiplied, each to form a streptomycin-resistant colony. Mutants with resistance to
streptomycin were being selected from the predominantly streptomycin-susceptible algae.
About 90 percent of the mutant involved nuclear genes (sr-1), and such mutations (sr-2),
however, were uniparental and nonchromosomal. Eventually, nonchromosomal mutants were
recovered from almost every colony. Nonchromosomal DNA mutations expressed the same
phenotypes as chromosomal genes are presumed to be located in the chloroplast.

Recripocal crosses demonstrated that antibiotic, resistance, controlled by

nonchromosomal genes, was uniparental in inheritance. On the other hand, mating type in this
sexual unicelular alga was controlled by chromosomal genes, which were designate 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 (+ famale)
mating type was resistant all progeny were resistant, when the plus (+) mating type was
nonresitant, all progeny were nonresistant. These result of reciprocal crosses demonstrated non-
mendelian inheritance, which involved a single pair of contrasting traits. Nonchromosomal
genes sr for streptomycin resistance and ss for streptomucin sensistive, were postulate to
control these two alternative characteristics.

Another mutant, ac2 which bloked photosynthetic activity, was induced and a pair
nonchromosomal alleles ac1 and ac2 was thus avaible for study in the same strain of
chlaydomonas. The mutant required acetace in the medium for growth. Wiyh two pairs of
nonchromosomal genes avaible a dyhibrid cross could be conducted in the sam e system to
check for evidence of recombination. Crosses of the dyhibrid type ac1 ss x ac2 sr were prepared
and progeny were allowed to grow for a vegetative multiplications. Each cell was then
classified for its segregating markers both nonchromosomal and chromosomal (mating type
and other known to be chromosomal). Both the ac1/ac2 and sr/ss pairs of alleles were observed
to segregate but not always in the same division. Afther four or five mitotic doublings both
parentals and recombinants had been obtained. The result indicated independent assortment
suggesting that the two pairs of nonchromosomal genes were carried in different plastids. Three
and four point crosss and reciprocal crosses have been made with the addition of several
mutant. Which are presumed to be carried in chloroplast and mitochondria. A genetic map of
nonmendelian genes in chlamydomonas has been constructed but uncertainly exists as to
whether some chloroplast linkage groups are solely in the chloroplast genome.

ORGANIZATION OF PLASTID GENOMES

The plastid genomes of over 200 species of higher plants and of many green, blue-green
and red algae have been al least partially characterized. Eithin a given species the genomes od
the different types of plastid chloroplast, amyloplast (plastid that accumulate starch is storage
tissue) and chromoplasts (plastid containing pigment) all are identical in organism where they
have been studied. Thus our discussion of plastid genome structure will be resstricted of the
organization of the DNAs of chloroplast (cpDNAs) the most important member of the plastid
family.

In higher plants, cpDNAs range in size from 120 to 160 kb. In algae, the size range for
chloroplast genomes is much larger from 85 to 292 kb for species known to have circular
cpDNAs. In two species of green algae of the genus acetabularia, the cpDNAs appear 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 about
100 copies of the cpDNA. The large single chloroplast of chlamydomonas reinhardiii contains
about 100 copies of the cpDNA. The single celled flagellate Euglena gracilis contains about 15
chloroplasts each with about 40 copies of cpDNA giving a total of about 600 copies per
organism.

All the chloroplast genomes analyzed to date contained basically the same set of genes
but with these genes arranged in very different ways on the cpDNAs. The genes presents on
cpDNAs can be grouped into two major classe : 1. Those that encode compenents of the
chloroplast protein biosynthetic apparatus (RNA polymerase subunits, structural components
of chloroplast ribosome and a set of RNA) and 2. Those specifying components of the
photosyntetic machinery (photosystems I and II and the electron transport chains).

Chloroplast genomes of higher plants are about one twentieth to one thirtieth the size
of the genomes of the prokaryotic organism frpm which they are believed to have evolved.
Thus the chloroplast have 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 componens are synthesized on cytoplasmic ribosome and
are imported into chloroplast with 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

about evolutionary relationships of plant and algal species. J.D Palmer has distinguished six
major lines of chloroplast evolution. The chloroplast genomes prsent in different evolutionary
lines all contain largely the same genes, but these genes are present in different arrange ments
of the cpDNA molecules. the rRNA genes are present in duolicated on inverse repeats within
the cpDNAs of most species. However, in euglena gracailis the 16S and 23S rRNA 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 genomes
palmer has been able to show that many of the changes in cpDNA organization have resulted
from inversions of segments of DNA were found to have occured in intergenic regions and
within introns of genes. However the major documented changes in cpDNA structure seem to
have resulted from large inversions.

Photosynthesis occuring within chloroplast provides the life sustaining energy souces
for all living organism on planet earth. Since chloroplast genomes encode many key
components of photosystems I and II and the electron transport chains, knowledge of the
structure and function of cpDNAs is very important and has received much attention. The
complete nucleotide pair sequences of the cpDNAs of the liverwort marchantia polymorpha
and of tobacco have been determined. The cpDNAs of marchantia and tobacco are 121, 024
and 155,844 nucleotide pairs in length, respectively. The organization of the single copy genes
in the chloroplast genomes of these two plants is remarkably similar considering that they are
evolutionarily very distant from each other. The major difference between these two cpDNAs
is that the inverse repeat regions contaoning the rRNA genes are considerably larger in tobacco.
The best estimates of cpDNA gene number are 136 in marchantia and 150 in tobacco. The
loctions 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 products that they
encode will have important practical applications in the future. Information of the exact
mechanisms by which photosystems I and II function might someday permit scientists and
engineers to build a totally synthetic system capble of duplicating the capacity of green plants
to capture light energy and convert it to chemical forms useful to living organisms.