BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 29. EUKARYOTIC CHROMOSOMES AND GENE EXPRESSION IN EUKARYOTES

29.12. Ribosomal RNA Genes Are Arranged in Tandem Repeats and Present in Hundreds of Copies

Genes encoding ribosomal RNA molecules possess two distinctive features. First, they are arranged in tandem repeats, with nearly all eukaryotes harboring over 100 copies of these genes. Second, the majority of rRNA genes reside in specialized chromosomal regions associated with nucleoli. Mutant Cells lacking nucleoli synthesize very little rRNA and are consequently non-viable. Much is known about these genes, largely due to the pioneering work of Mac-

by Max Birnstiel, Donald Brown, Oscar Miller, and their colleagues. The genes encoding the four Ribosomal RNAs—18S, 5.8S, 28S, and 5S rRNA—were isolated in pure form from the DNA of the African clawed frogs Xenopus laevis and Xenopus mulleri. The clawed frogs were chosen because their oocytes contain an unusually high Abundance of these genes.

The 18S, 5.8S, and 28S rRNA genes are clustered together and repeated in tandem. In situ Hybridization demonstrated that these genes are localized in the nucleoli. The repeating unit consists of a Gene encoding a 40S RNA precursor and a spacer (Fig. 29.21). The 40S precursor (8kb) is modified and cleaved to yield mature 18S, 5.8S, and 28S rRNAs (Sec. 29.21). The spacer (non-transcribed region) is not transcribed (Fig. 29.22). Somatic frog cells contain approximately 500 copies of this repeating unit, all arranged consecutively. During oogenesis, a remarkable selective Amplification (increase in copy number) of these genes takes place. They replicate several thousand times to yield roughly 2 • 10⁶ copies. The rRNA-encoding genes account for up to 75% of the total oocyte DNA. The amplified DNA exists as extrachromosomal circles attached to numerous de novo-formed nucleoli. Such selective gene amplification enables oocytes to accumulate the 10¹² Ribosomes required for extremely rapid Protein Synthesis during Cleavage. Without gene amplification, The production of 10¹² ribosomes would take several centuries!

Class="center">Fig. 29.21. Organization OF THE 40S precursor genes for 18S, 5.8S, and 28S rRNAs in Xenopus. The tandemly repeated genes are separated by non-transcribed spacers. The length of the repeating unit is 13 kb

Fig. 29.22. This electron micrograph of nucleolar DNA clearly reveals the tandem arrangement of the 18S, 5.8S, and 28S rRNA genes. The thick axial strand represents DNA. The thin lateral fibrils extending from the axis are newly synthesized RNA molecules complexed with Proteins. THE ORIGIN OF each "Christmas tree" Structure, formed by the transcribing RNA molecules, corresponds to the METABOLISM/31.html">Transcription initiation site. The bare stretches between the tree origins are non-transcribed spacers

The gene encoding the smallest ribosomal RNA molecule, 5S rRNA, also occurs in tandem repeats. Both somatic cells and oocytes contain approximately 24,000 copies of this gene, which encodes an RNA molecule 120 NUCLEOTIDES in length. These genes are organized into clusters located at the ends of most frog Chromosomes. In this case as well, a non-transcribed spacer region is found between the rRNA genes (Fig. 29.23). In fact, the spacer is several times longer than the gene itself. Interestingly, it contains a non-transcribed pseudogene whose base sequence resembles the gene itself. The function of the spacer in this and other eukaryotic genes remains an enigma.

29.13. Histone genes are clustered and repeated in tandem many times

How are protein-coding genes organized? Histone genes were among the first to be isolated and characterized, thanks to the abundance of histone mRNA in rapidly dividing sea urchin embryos. These marine invertebrates develop from the zygote stage to a 1,000-Cell blastula stage in just 10 hours. Consequently, assembling new Chromatin requires the synthesis of massive amounts of histone. Indeed, during early Embryogenesis, Histones account for more than a quarter of all synthesized protein. Histone mRNA is even more abundant, making up about 70% of all messenger RNAs synthesized at this stage, which makes it relatively easy to isolate. Once isolated, the hybridization kinetics of histone mRNA with sea urchin DNA were studied to determine the copy number of histone genes. The hybridization rate was several hundred times higher than expected for unique sequences, indicating that histone genes are characterized by a very high degree of repetition. The copy number of histone genes in various sea urchin species ranges from 300 to 1,000. In other organisms, repetition is lower (Table 29.4). The copy number of histone genes in a given Organism apparently correlates with the demand for rapid histone mRNA synthesis.

Fig. 29.23. Organization of 5S rRNA genes in Xenopus laevis. These tandemly repeated genes are also separated by non-transcribed spacers. The repeating unit is approximately 750 Base Pairs long

Fig. 29.24. Light micrograph of a sea urchin embryo at the four-cell stage

Table 29.4. Repetition frequency of histone genes¹

How are multiple copies of histone genes organized within The Genome? To obtain a DNA fraction enriched in histone genes, researchers exploited the fact that these genes contain a higher G-C base content (55%) than the bulk of sea urchin DNA (42%) and, consequently, exhibit a higher buoyant density. Digestion of

such enriched DNA with a restriction endonuclease yielded 7 kb fragments that hybridized with histone mRNA. These fragments were then cloned in E. coli cells and analyzed using restriction Enzymes and Electron Microscopy. The results showed that the genes encoding the five major histones are grouped together within a basic repeating unit 7 kb in length (Fig. 29.25). Within this repeating unit, five coding regions alternate with five spacers. Unlike other eukaryotic genes described previously (Section 26.12), the coding sequences are not interrupted by intervening sequences. The cluster of five histone genes is repeated in tandem many times. The repeats are very similar to each other, though not identical. This is consistent with the fact that histones H1, H2A, and H2B represent families of closely related proteins rather than entirely homogeneous Polypeptides. Different histone genes are expressed in various Tissues and at different developmental stages.

¹ The Myosin gene contains over 50 introns! — Transl. note.

Fig. 29.25. Map of clustered histone genes in the sea urchin (Strongylocentrotus purpuratus) and fruit fly (Drosophila melanogaster). Coding regions are shaded in blue, and spacers (non-transcribed regions) in yellow. Arrows indicate the direction of transcription

What is the functional role of this particular Introduction/29.html">Gene Organization? Their reiteration is undoubtedly crucial for the rapid synthesis of large amounts of histone mRNA. However, the reason why these repeats are arranged in tandem is less clear. The close linkage of the genes encoding all five histones may play an essential role in coordinating their expression, ensuring that histones H2A, H2B, H3, and H4 are produced in equimolar amounts, while histone H1 is synthesized at half that level.

29.14. Many Abundantly Synthesized Proteins Are Encoded by Unique Genes

We have already seen that the genes for ribosomal RNAs and histones are reiterated many times. Are genes that encode large quantities of a product generally represented by multiple copies? To address this question, Donald Brown isolated the gene for Silk Fibroin from the silkworm Bombyx mori. This system was chosen because the mRNA encoding silk fibroin possesses distinctive structural features, serving as a template for repetitive Amino acid sequences rich in Glycine. Furthermore, giant cells of a single specialized cell type synthesize massive amounts of silk fibroin at a specific developmental stage. The RNA was purified taking advantage of its large size (9,1 kb) and high G content relative to rRNA and other RNA molecules. The silk fibroin gene was also partially purified thanks to its unusually high buoyant density. Hybridization of purified fibroin mRNA with DNA revealed that the haploid genome contains only a single silk fibroin gene.

Fig. 29.26. Silkworm

This finding was of paramount importance for The Study of EUKARYOTIC Gene Expression and differentiation. It demonstrated that abundant quantities of a specific protein can be synthesized even when the genome contains only a single copy of its coding gene. A single silk fibroin gene serves

as a template for the synthesis of approximately 10 mRNA molecules that persist for several days. Each mRNA molecule, in turn, serves as a template for the production of about 10 protein molecules. Thus, a single gene is sufficient to direct the synthesis of roughly 10 protein molecules over a four-day period. It appears that the reiterated genes for ribosomal RNAs and histones are exceptions rather than the rule. A single silk fibroin gene is far more typical of protein-coding genes, even those encoding abundant proteins. For example, reticulocytes contain one or a few copies of the genes encoding Hemoglobin subunits (Section 29.26). Similarly, massive amounts of Ovalbumin—the major component of egg white—are synthesized in the oviduct of laying hens, where cells contain only a single copy of the ovalbumin gene per haploid genome. Nevertheless, one should not overlook the possibility of selective gene amplification as a mechanism for enhancing the Synthesis of specific proteins in certain cell types. Some cultured tumor cells acquire resistance to Folic acid analogs by synthesizing up to 200-fold more Dihydrofolate Reductase than normal sensitive cells. This elevated enzyme level results from the selective amplification of the dihydrofolate reductase gene. Clearly, the eukaryotic genome is a remarkably dynamic system.