In this article we will discuss about:- 1. Introduction to the Kinds of Genes in Eukaryotes 2. Repetitive DNA Sequences 3. Transcription of DNA and RNA in Eukaryotes 4. Mitochondrial DNA and RNA.
Introduction to the Kinds of Genes in Eukaryotes:
The principle of “one gene, one protein” does not apply to all genes. There are genes which do not code for any proteins at all, although they perform a most important function in cells of all eukaryotes and some prokaryotes. Among these are the genes coding for RNA’s that are not translated into protein, namely for the ribosomal RNA’s (rRNA’s) and the transfer RNA’s (tRNA’s). There are thus at least three very different groups of genes – rRNA genes, tRNA genes and genes coding for messenger RNA (mRNA).
a. Ribosomal RNA Genes:
The ribosomes are the complex cell organelles inside of which the message contained in the mRNA is translated into protein structure. Ribosomes are constructed from a number of RNA molecules and protein molecules. There are three kinds of RNA molecules – the 28S, the 18S and the 5S molecules.
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The figures stand for the different rates, in “Svedberg” units, at which the molecules are sedimented in a centrifuge. The higher the number, the faster the molecules sediment; this in turn indicates a greater mass or, in other words, a higher molecular weight. The three kinds of molecules are made up of approximately 4700, 2200 and 120 nucleotides, respectively.
The ribosomal genes are present in eukaryotic cells in a large number of copies per cell. In the cells of the frog Xenopus laevis there are approximately 900 copies each of the 28S and 18S genes and about 28,000 copies of 5S genes per cell. Furthermore, all the 28S and 18S genes are concentrated in one section of a particular chromosome, which forms a nucleolus in cells in interphase.
The 5S genes are present in practically all chromosomes, located at their ends. Because the ribosomal genes are present in large numbers per cell, they can be separated and purified by direct methods, e.g., breaking up the total DNA and subdividing it into fractions by ultracentrifugation.
The codes for the 28S and the 18S RNA’s are contained in two genes lying close together in one of the repetitive units of the nuclear DNA. These two chromosomal genes are separated by a short “spacer,” and between each pair of genes there is a longer spacer sequence.
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This arrangement of the two ribosomal RNA genes has been found in all eukaryotes studied, from yeast cells to higher plants and mammals. The two ribosomal genes are transcribed together into a long molecule with a sedimentation rate of 40S. In addition to the ribosomal RNA proper this long molecule also contains transcripts of the small spacer between the two genes, and a part of the larger spacer adjacent to the 18S gene, but does not contain a transcript of the greater part of the long spacer between the gene repeats.
Subsequently the small spacer between the two genes as well as the section complementary to the small part of the long sequence adjacent to the 18S gene is cut off. In this way about 20 per cent of the original transcript is discarded, and the 28S and the 18S RNA molecules are brought to their final composition.
The 5S ribosomal genes are also arranged in repetitive units, the genes being separated by quite long spacers. The spacers are of different lengths in different species – from about 6 times the length of the 5S gene in Xenopus laevis to 18 times the length of the gene in Xenopus mulleri.
The 120 nucleotides constituting the 5S gene are identical in all mammals but may show some variation in other animals, and there can be several variations in one and the same animal, as in Xenopus laeuis, where 5S rRNA in the oocyte differs from 5S rRNA in body cells.
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The ribosomal genes do not make up a very large proportion of the chromosomal DNA. The situation in the oocyte, is a special case. In ordinary somatic cells, the DNA concerned with the production of the ribosomal RNA makes up no more than 0.15 to 0.2 per cent of the total chromosomal DNA.
b. DNA Coding for Transfer RNA:
The transfer RNA’s are the smallest polynucleotides, making them the easiest objects for complete sequencing. In fact, most tRNA’s have been completely sequenced. In the case of tRNA, the section of DNA involved in its production is also larger than the eventually formed RNA. The tRNA for tyrosine consists of 85 nucleotides, but is synthesized originally as a precursor consisting of 126 nucleotides.
Forty-one nucleotides are split off from the precursor before the final functional molecule is ready. The gene which produces tyrosine transfer RNA, in addition to the sequence coding for the whole precursor molecule, also consists of an “initiator” sequence of unknown length and a “terminator” sequence of at least 24 nucleotides.
The remarkable discovery arising from the work of Korana and coworkers is that tRNA’s are very similar in all organisms, from bacteria to mammals and higher plants. This would suggest that the genes coding for the tRNA’s are also similar in eukaryotes and prokaryotes. This is, of course, not necessarily so.
In fact, it has been found that some eukaryotic tRNA genes have intervening sequences which are not reproduced in the tRNA itself. Thus, yeast genes for tyrosine and phenylalanine tRNA’s have such intervening sequences (of 14-20 nucleotide pairs) close to the nucleotides coding for the anticodon group. Such intervening sequences or spacers are characteristic of eukaryotic genes but are very exceptional in prokaryotic genes.
Transfer RNA genes, like the ribosomal genes, are present in a eukaryotic genome in a very large number of repeats (between 2000 and 13,000) per cell.
c. Structural Genes (Genes Coding for mRNA):
The genes coding for mRNA (and thus subsequently for proteins) are perhaps of the greatest interest for embryologists, as on these genes depends the diversification of the cells and tissues of the developing embryo.
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The genes coding for histones are in some respects a separate group, differing from genes coding for other proteins. All five histone genes are joined in a repetitive sequence which is reiterated a number of times – from about 10 to 20 times in the mouse, 30 to 40 times in man, 110 times in Drosophila.
Within each sequence the five genes are separated from each other by “spacers” who are not transcribed into messenger RNA.While the coding sequences for the genes are extremely similar in different organisms, the spacer sequences may differ very much even between closely related animals.
The transcribed sequence of each gene consists of three parts – the actual code for the histone protein, a “leader” sequence preceding the histone code, and a “trailer” sequence following the histone code. The leader and trailer sequences are represented in the mRNA, but are not translated into protein (histone) structures.
When the histone mRNA is synthesized, a portion of the spacer sequences is also transcribed, but lost later, before the mRNA attains its final structure. It appears that although the histone genes are located close together on the chromosome, each gene is transcribed into mRNA separately from the others.
It has been noted that all five histone molecules are present in the unit segment of chromatin (nucleosome + linker) in equal numbers. It is interesting, therefore, that the synthesis of the histones in equal quantities is provided by all five histone genes being present in each of the reiterated sequences on the chromosome.
One of the most spectacular differences between the genes coding for mRNA in eukaryotes and those in prokaryotes consists in interruptions frequently present in the eukaryotic mRNA genes—in the presence within a gene of sequences that are not represented in the mRNA derived from the gene. This contradicts the “principle of co-linearity” prevailing in prokaryotes, according to which the sequence of ribonucleotides in the RNA corresponds, nucleotide by nucleotide, to the sequence of deoxyribonucleotides in the gene DNA.
The best known examples of “interrupted” genes are the genes coding for β-globin (one of the protein components of hemoglobin), and for ovalbumin in the oviduct of a laying hen. Inside of a β-globin gene in both the rabbit and mouse there is a spacer or “intron” with a length of 600 nucleotide pairs (in the rabbit) or 550 nucleotide pairs (in the mouse).
The spacer exceeds by far the length of the meaningful part of the gene or “exon,” which is 146 nucleotide pairs in the rabbit. The ovalbumin gene is even more fragmented – it is split into seven separate, meaningful sequences (exons), separated by six spacers (introns), which are not represented in the ovalbumin mRNA.
The presence of spacers within a gene is best demonstrated by hybridizing the chromosomal gene extracted directly from cells with the corresponding mRNA. In such hybridization experiments the nucleotides of the meaningful sequences of the chromosomal genes find their counterparts in the nucleotides of the mRNA, but the nucleotides of the spacer sequences have no counterparts in the mRNA.
As a result, pairing occurs only between the meaningful section of the chromosomal DNA and the mRNA, while the spacer sections of the chromosomal DNA “loop out” from the hybrid molecules. The “looping out” sections can be seen easily when the hybrid molecules are examined with the electron microscope. Complicated “splitting” is found to occur also in the DNA sequences of genes coding for immunoglobulins.
It is rather remarkable that the split gene phenomenon has been detected in an animal virus, SV40, which as a prokaryote should have adhered to the “principle of co-linearity”; however, infecting animal cells, as it does, it seems to have adopted the construction of the animal genomes, with spacer sequences included in its DNA.
A second difference between eukaryotic genes and those of prokaryotes consists in the absence in eukaryotes of the standard “initiation sites” of prokaryotic genomes. In prokaryotes transcription of a gene always starts with the triplet coding for methionine.
Not all methionines are, however, starting points for transcription, but only those that are preceded by a short sequence of nucleotides, similar but not exactly the same for every gene. No such sequences have been detected in the case of eukaryotic genes. Instead, it is found that certain, sometimes quite lengthy leader sequences precede the beginning of the actual coding sequence of the gene.
The leader sequences are different in different genes and are included in the initial transcript from the DNA. The leader sequences are transcribed, but not eventually translated into polypeptide sequences. Leader sequences are also present in animal viruses.
In prokaryotes all genes are presumed to be present in a single copy. We have seen that rRNA genes in eukaryotes are represented by very large numbers of copies. It is presumed that mRNA genes in eukaryotes are present in single, or in only a few copies per haploid set of chromosomes, though a priori it is not possible to assert that all mRNA genes are thus represented.
Repetitive DNA Sequences:
The genes in the DNA enumerated so far do not by any means constitute the whole of the chromatin in a eukaryotic cell. If the DNA of such a cell is broken up into fragments of varying length, and the fragments are allowed to hybridize, it is found that there are numerous fragments that hybridize quickly, which means that they can easily find corresponding (complementary) partners.
This in turn means that there are large numbers of repetitive (identical) DNA sequences in the genome. Some, but not all, of these repetitive sequences are the ones coding for rRNA and tRNA. There are very large numbers of short (200 to 500 nucleotides in length) sequences, the function of which is not known at present.
In the rat genome it has been claimed that 70-75 per cent of the DNA is in unique sequences (presumably coding for mRNA), that 15-20 per cent of the DNA is repeated 10 to 100,000 times (the rDNA and tDNA genes fall into this category), and that 6 per cent of the DNA consists of sequences that are repeated more than 100,000 times.
There is good reason to suspect that these repetitive sequences serve somehow to control and regulate the functioning of the genes of the genome. Furthermore, there is a special class of eukaryotic DNA that in re-association experiments tends to fold back upon itself and form double-stranded hairpin loops.
This is the result of the presence in the DNA chromatin of base sequences that are arranged in a reverse order (“palindrome” sequences, like words and sentences which read the same back to front). There is some evidence that these loops provide recognition sites in some nucleic acid-protein interactions.
Finally, very short (6-15 nucleotides in length) repetitive DNA sequences, which are neither transcribed nor translated, are grouped in enormous numbers repeated 103 to 107 times in the centromere region of eukaryotic chromosomes.
Transcription of DNA and RNA in Eukaryotes:
Transcription of the DNA into RNA in eukaryotes is performed by a different set of enzymes than in prokaryotes. In prokaryotes the DNA polymerase extracted from Escherichia coli cells appears to be the universal tool of transcription. In eukaryotes there are several polymerases—polymerases A, B, and C—which have a different action from the bacterial polymerase, and from each other.
The differences between the polymerases are shown in their sensitivity to the antibiotic α-amanitin (the mushroom poison), and also in their ability to transcribe certain kinds of genes. Polymerase A (or I) is insensitive to α-amanitin even in rather high concentrations (200 mg/ml). It transcribes the genes for 28S and 18S ribosomal RNA’s.
Polymerase B (or II) is highly sensitive to α-amanitin, being inhibited by 1 µg/ml of that poison. It transcribes the genes coding for proteins (for mRNA). Polymerase C (or III) is slightly sensitive to α-amanitin. It transcribes the genes for 5S rRNA and for tRNA.
Mitochondrial DNA and RNA:
Mitochondrial DNA:
In addition to the genes which are contained in the chromosomes of the nucleus, eukaryotes possess DNA molecules outside the nucleus, in the mitochondria. Each mitochondrion contains a small “chromosome,” which is active in being transcribed into messenger RNA and controls the synthesis of proteins.
In the mitochondrial chromosome the DNA is not associated with histones, and the chromosome is thus a naked DNA molecule. In this respect it resembles the chromosomes of prokaryotes (bacteria and viruses). Also, like the bacterial chromosome, the mitochondrial chromosome is closed into a ring.
The amount of DNA in a mitochondrial chromosome is infinitesimally smaller than the amount of DNA in the nucleus; it is less than the amount contained in the smaller viruses (molecular weight about 11 million). The number of nucleotide pairs is sufficient for 10-25 average-sized genes.
The chemical mechanisms in the mitochondrial chromosome are also different from those in nuclear chromosomes. The antibiotic rifampicin prevents transcription in mitochondria and in bacterial cells, but does not affect transcription in eukaryotic nuclear chromosomes. On the other hand actinomycin, which inhibits transcription in eukaryotic nuclear DNA, does not inhibit mitochondrial transcription.
The similarities between the mitochondrial chromosomes and those of bacteria and the differences from eukaryotic nuclear chromosomes are so striking that the suggestion has been made that mitochondria were originally independent bacteria-like organisms, which became endosymbionts of other cells—the cells giving rise to eukaryotes. According to this theory, in the course of further evolution the endosymbionts would have lost much of their independence.
At present, the synthesizing activities of the mitochondria are very limited. The production of the mitochondrial ribosomes is definitely the result of action of the mitochondrial genes; in addition, these genes control apparently only some proteins involved in the structure of the mitochondrion. The rich systems of enzymes contained in the mitochondria, in particular the system of oxidative enzymes, are produced by nuclear chromosomal genes.
Mitochondrial RNA:
In proportion to the small size of the mitochondrial chromosomes, the amount of RNA synthesized in the mitochondria is very minute compared to the RNA synthesized in the nucleus (in spite of the large number of mitochondria per cell).
Only during early cleavage, when the nuclear genome has not yet been multiplied by repeated mitosis, the amount of RNA synthesis in the mitochondria may be relatively quite considerable – in sea urchin embryos in early cleavage up to 50 per cent of all newly synthesized RNA is of mitochondrial origin.