Chloroplast genetics Non-chromosomal genes in Chlamydomonas

In Chlamydomonas, although the first non-Mendelian gene controlling streptomycin resistance was described by Ruth Sager (a lady scientist in U.S.A.) as early as 1954, but its location on chloroplast genome was known later. Several gene pairs located on chloroplast genome were reported and a circular genetic map was proposed by Sager in 1972 in her book "Cytoplasmic Genes and Organelles". Later she presented further results and discussed methods used in the genetic analysis of these non-Mendelian genes.

(a) Uniparental inheritance.

As shown earlier, several characters show maternal inheritance both in plants and animals. This was earlier explained to be due to greater amount of cytoplasm in egg than in sperm or male gamete. In Chlamydomonas, which is a haploid organism, sexual reproduction is isogamous, both gametes being similar in morphology and thus having equal amounts of cytoplasm. Despite this lack of difference in cytoplasmic content, in Chlamydomonas, some characters are transferred only from one parent, exhibiting what is called uniparental inheritance, rather than maternal inheritance, because in Chlamydomonas, we describe two sexes as two mating types, i.e., mt⁺ (plus) and mt^- (minus), rather than as female and male (Fig. 18.16). One may notice that in diploids, the male gametes do not contribute cytoplasm (Fig. 18.17). One may also distinguish between the patterns of inheritance between non-chromosomal and chromosomal genes. The inheritance patterns for chromosomal genes in haploid and diploid organisms are shown in Figures 18.18 and 18.19.

Uniparental inheritance has also been demonstrated at the level of cpDNA, due to the following facilities : (i) cpDNA forms a separate distinct band on CsCl density gradient centrifugation (Fig. 18.20); (ii) the cpDNA of two parents can be differently labelled with N¹⁴ and N1⁵, the two giving buoyant densities of 1.69 and 1.70 respectively; this difference looks small, but they form separate bands on CsCl density gradient. It was shown that the buoyant density of zygote always resembled that of mt⁺ (female) parent and that cpDNA of mt^- parent is lost, inactivated or destroyed. Restriction enzyme patterns of cpDNA in zygote also resembled those of mt⁺ parent suggesting uniparental transmission of cpDNA.

(b) Segregation, independent assortment and gene mapping.

In earlier years (before 1960), a large number of cases were known, where non-chromosomal inheritance, as evident from uniparental or maternal inheritance, were described. However, further detailed study of these traits was not possible. There were two reasons for this. First, the mutant non-chromosomal genes could not be artificially produced when desired and spontaneous mutations for these genes were rare, thus making their study difficult. Second, since these traits were transmitted from only one parent, standard techniques of genetic analysis could not be applied to study segregation and independent assortment as studied in chromosomal genes. Ruth Sager was successful in overcoming both these difficulties in Chlamydomonas and was thus able to study the non-chromosomal genes in this organism in some detail.

Ruth Sager found that if streptomycin sensitive cells are grown in a medium containing streptomycin, several kinds of induced mutations in chloroplast including streptomycin resistant mutations were produced. Streptomycin resistance could be found on chromosomes also where it may be inherited in a Mendelian manner, but in some strains this character showed uniparental inheritance as shown in Figure 18.21. It may be noticed that when the mt⁺ strain was streptomycin resistant, all the progeny was streptomycin resistant and when it was sensitive, all the progeny sensitive. Streptomycin was used for inducing not only resistance to streptomycin but also to induce mutations for several other non-chromosomal traits thus making it possible to study their inheritance.

The second difficulty of getting the transmission of non-chromosomal genes from only one parent was overcome when it was observed by Ruth Sager that in very rare cases streptomycin resistance and streptomycin sensitivity from parents of both mating types, mt⁺ and mt^- could be transmitted. It was found that, when streptomycin dependent strain (sd, which will not survive without streptomycin) was used as mt⁺ strain, and progeny grown in a medium containing streptomycin, only sd strains will be produced (Fig. 18.22 A). However, if the same progeny is grown in a medium lacking streptomycin, then biparental exceptional zygotes will grow, because the uniparental sd strains will not survive in the absence of streptomycin (Fig. 18.22 B). Similarly in the reciprocal cross, using ss (streptomycin sensitive) as the mating type mt⁺ and sd as mt^-, only ss strains will be available in the absence of streptomycin showing uniparental inheritance (Fig. 18.22 D), but, if the progeny is grown in the presence of streptomycin, then biparental zygotes will be produced (Fig. 18.22 C). Later it was discovered that if mt⁺ parent is irradiated with ultraviolet (UV) irradiation prior to mating, frequency of biparental zygotes will greatly increase. In this manner, techniques for obtaining exceptional biparental zygotes could be standardized.

As shown above, Ruth Sager could overcome the two difficulties for genetic analysis of chloroplast genes. After having achieved this, Sageri and her co-workers made crosses between strains carrying contrasting characters controlled by non-chromosomal genes. This will be illustrated with the help of an example used by her in her article published in Scientific American in 1965. The mt⁺ parent carried ac₁ (acetate requiring mutant) and sd (streptomycin dependence), and mtr parent carried ac₂ and sr (the other alleles of same genes ac₁ and sd). The parents also differed in two other nuclear genes, mt⁺ carrying nuclear genes act-r (antibiotic actidione resistance) and ms-r (resistance to methionine sulfoximine), and the mt^- strain carrying nuclear genes, acts (actidione sensitive) and ms-s (sensitive to methionine sulfoximine). The nuclear genes (which were not linked) were used as markers to ensure that zygotes were produced and segregation for them was according to expectation.

It was found that 99 per cent progeny though segregated for nuclear genes act-r/act-s, ms-r/ms-s and mt⁺/mt^- but they all showed maternal or uniparental transmission of ac₁ and sd. One per cent exceptional biparental zygotes were obtained, which were selected and their behaviour in future generations was studied to explain the pattern of inheritance of non-chromosomal genes. The details of the results are shown in Figure 18.23. It was shown that during meiosis the nuclear genes segregated, but non-chromosomal genes did not segregate immediately, and segregated slowly in the following generations. The following conclusions were made on the basis of the results shown in Figure 18.23.

1. The non-chromosomal or chloroplast genes segregate like nuclear genes in 1 : 1 ratio in mitotic divisions, so that ac, and ac₂ as well as sd and sr behaved as alleles.
2. The ac₁/ac₂ and sd/sr gene pairs segregate independently of each other, in both time and space, giving rise to parental combinations ac₁ sd and ac₂ sr as well as the recombinants ac₁ sr and ac₂ sd.
3. Even wild type or new cytoplasmic alleles arose, which are designated as ac⁺, ac', ss, sd' and sr'. These new alleles are believed to have originated due to intragenic recombination discussed in Fine Structure of Gene-at the Genetic Level (A New Concept of Allelomorphism) for nuclear genes.

Several other non-chromosomal genes were studied using the above and other techniques. Some of these genes include the following : (i) ac (acetate requiring mutant) with reduced ability to grow and giving small flat colonies; (ii) Sm², streptomycin resistance; there is another gene symbolised as sd, which confers streptomycin dependence, so that the organism is not only resistant, but is dependent and can not grow without the drug;- (iii) ery, erythromycin resistance; (iv) spc, spectinomycin resistance; (v) tm₁, temperature sensitive, which grows only at 25°C and not at 35°C.

A circular genetic map of chloroplast genome of Chlamydomonas is shown in Figure 18.24, where an attachment point ap is also shown. (For details consult Ruth Sager's book).

Chloroplast genetics in higher plants.

We know that chloroplasts of lower eukaryotes and higher plants like Zea mays contain DNA. Chloroplast genomes are relatively large (with a narrow range) usually ranging from 120 kb to 210 kb (kb = kilo base pairs) except in Acetabularia (400 kb). This is comparable to the size of a large bacteriophage like T4 (110 kb). Each cell may have several chloroplasts and each chloroplast may have several circular DNA molecules, so that the chloroplast DNA (cpDNA) may make upto 14% of cellular DNA in lower eukaryotes. In higher eukaryotes, however, the cpDNA makes a small proportion of total DNA in the cell. These features of chloroplast DNA are presented in Table 18.1.


There is evidence that genes controlling specific traits or those controlling the synthesis of specific molecules of RNA and protein, are found on the chloroplast DNA. The presence of these genes could be shown by a technique called restriction fragment analysis or restriction site mapping (see Genetic Engineering and Biotechnology 2.  Restriction Maps and Molecular Genetic Maps). In this technique, using specific enzymes, the endonucleases, also called restriction enzymes (enzymes which break DNA at specific sites), DNA can be cut into fragments. Each enzyme will cut the DNA into a definite number of segments of definite sizes and the different enzymes differ in their effect. Using different restriction enzymes and through an analysis of fragments produced, sites could be mapped on chloroplast genome. The mapping of specific genes was also facilitated by hybridization of restriction fragments with RNA transcripts, produced in the cell and suspected to have originated on chloroplast genome. In this manner, small and large rRNA (ribosomal RNA) could be shown to be synthesized by specific genes on chloroplast DNA (cpDNA). In maize, the sequence of rRNA gene is shown to be duplicated. Similarly genes for tRNA (transfer RNA) have also been mapped.

The genes on cpDNA synthesize not only rRNA and tRNA, but also some proteins through the synthesis of mRNA (messenger RNA). Ribulose bisphosphate carboxylase (rubisco) is one such important enzyme, which is abundant in green leaves. Each molecule of this enzyme has 16 polypeptides, eight small (100 amino acids long) and eight large (450 amino acids long). Although small subunit is synthesized through nuclear genes, thebigger is under the control of cpDNA. The map of maize chloroplast genome known at present is shown in Figure 18.25.

Chloroplast genome and prokaryotes (a comparison).

A detailed study of chloroplast genome in eukaryotes, has revealed that the chloroplast genomes share both prokaryotic and eukaryotic features. Therefore, the view that they are the remnants of entrapped prokaryotic genomes is an oversimplification. Some of the features in which chloroplasts resemble the prokaryotes and those in which they differ are as follows—(for more details consult Expression of Gene : Protein Synthesis 4. Translation in Prokaryotes and Eukaryotes).

(a) Resemblances between chloroplast and prokaryotes.

1. The ribosomes are 70S in size in both the cases.
2. The rRNA genes as well as several tRNA genes have very high degree of homology as revealed by sequence homology.
3. Preceding the initiation codon AUG in chloroplasts, there are present ribosome binding sites, and the sequences which bear strong resemblance to 'Pribnow Box' (-10 and the -35 regions) of bacteria (consult Expression of Gene : Protein Synthesis 2.  Transcription in Prokaryotes and Eukaryotes and Regulation of Gene Expression 1.  Operon Circuits in Bacteria and other Prokaryotes).
4. There is sequence overlapping between some genes in chloroplasts, a feature known in prokaryotes. In chloroplasts it can not be due to economy since non coding sequences are also available in chloroplasts.
5. There is a resemblance between RNA polymerases of chloroplasts and prokaryotes. For instance, in Chlamydomonas reinhardii the enzyme is sensitive to rifampicin and contains a a (sigma) like polypeptide. There is also evidence of homology between p and p' subunits of E. coli RNA polymerase and those of C. reinhardii chloroplast RNA polymerase.
6. Protein synthesis in chloroplasts can be inhibited by chloramphenicol as in bacteria, since only 70S ribosomes are sensitive to this drug. Cycloheximide does not inhibit protein synthesis in chloroplasts, as it does in the cytoplasm (SOS ribosomes) of eukaryotes.
7. Initiation of protein synthesis takes place by formyl methionyl tRNA both in chloroplasts and prokaryotes.

(b) Differences between chloroplast and prokaryotic genomes.

1. In chloroplasts there are split genes, e.g. (i) rRNA genes and protein genes in chloroplasts of C. reinhardii, (ii) protein genes in chloroplasts of Euglena and (iii) tRNA genes of several chloroplasts of higher organisms. The introns of these split genes fall in three groups and differ from those in nuclear genes of eukaryotes (consult Organization of Genetic Material 3.  Split Genes, Overlapping Genes and Pseudogenes).
2. Chloroplast DNA sequences have the ability to promote autonomous replication. This feature is absent in prokaryotes.
3. Presence of short repetitive elements interspersed throughout several, but not all chloroplast genomes is also a eukaryotic feature.