4.5S RNA is encoded by hundreds of tandemly linked genes, has a short half-life, and is hydrogen bonded in vivo to poly(A)-terminated RNAs in the cytoplasm of cultured mouse cells.

4.5S RNA is a group of RNAs 90 to 94 nucleotides long (length polymorphism due to a varying number of UMP residues at the 3' end) that form hydrogen bonds with poly(A)-terminated RNAs isolated from mouse, hamster, or rat cells (W. R. Jelinek and L. Leinwand, Cell 15:205-214, 1978; F. Harada, N. Kato, and H.-O. Hoshino, Nucleic Acids Res. 7:909-917, 1979). We have cloned a gene that encodes the 4.5S RNA. It is repeated 850 (sigma = 54) times per haploid mouse genome and 690 (sigma = 59) times per haploid rat genome. Most, if not all, of the repeats in both species are arrayed in tandem. The repeat unit is 4,245 base pairs long in mouse DNA (the complete base sequence of one repeat unit is presented) and approximately 5,300 base pairs in rat DNA. This accounts for approximately 3 X 10(6) base pairs of genomic DNA in each species, or 0.1% of the genome. Cultured murine erythroleukemia cells contain 13,000 molecules per cell of the 4.5S RNA, which can be labeled to equilibrium in 90 min by [3H]uridine added to the culture medium. The 4.5S RNA, therefore, has a short half-life. The 4.5S RNA can be cross-linked in vivo by 4'-aminomethyl-4,5',8-trimethylpsoralen to murine erythroleukemia cell poly(A)-terminated cytoplasmic RNA contained in ribonucleoprotein particles.     

Genome complexity of the maize rust fungus,Puccinia sorghi

The base composition and complexity of genomic DNA fromPuccinia sorghi have been estimated by thermal denaturation, analytical ultracentrifugation, and reassociation kinetics. The buoyant density of genomic DNA in CsCl was found to be 1.7021 g/ml, which corresponds to a GC content of 43%. From thermal denaturation curves the GC content was estimated to be 41%. The haploid genome size ofP. sorghi was estimated to be 4.7 × 10⁷ bp, half of which represented a moderately repetitive fraction. The size of theP. sorghi genome is similar to that of other basidiomycete fungi; however, the amount of repetitive DNA is greater than that reported for most other fungi.     

GC-biased gene conversion impacts ribosomal DNA evolution in vertebrates, angiosperms, and other eukaryotes

Ribosomal DNA (rDNA) is one of the most conserved genes in eukaryotes. The multiples copies of rDNA in the genome evolve in a concerted manner, through unequal crossing over and/or gene conversion, two mechanisms related to homologous recombination. Recombination increases local GC content in several organisms through a process known as GC-biased gene conversion (gBGC). gBGC has been well characterized in mammals, birds, and grasses, but its phylogenetic distribution across the tree of life is poorly understood. Here, we test the hypothesis that recombination affects the evolution of base composition in 18S rDNA and examine the reliability of this thoroughly studied molecule as a marker of gBGC in eukaryotes. Phylogenetic analyses of 18S rDNA in vertebrates and angiosperms reveal significant heterogeneity in the evolution of base composition across both groups. Mammals, birds, and grasses experience increases in the GC content of the 18S rDNA, consistent with previous genome-wide analyses. In addition, we observe increased GC contents in Ostariophysi ray-finned fishes and commelinid monocots (i.e., the clade including grasses), suggesting that the genomes of these two groups have been affected by gBGC. Polymorphism analyses in rDNA confirm that gBGC, not mutation bias, is the most plausible explanation for these patterns. We also find that helix and loop sites of the secondary structure of ribosomal RNA do not evolve at the same pace: loops evolve faster than helices, whereas helices are GC richer than loops. We extend analyses to major lineages of eukaryotes and suggest that gBGC might have also affected base composition in Giardia (Diplomonadina), nudibranch gastropods (Mollusca), and Asterozoa (Echinodermata).     

Studies on macronuclear DNA from Paramecium aurelia

Macronuclear DNA was isolated from purified macronuclei of Paramecium aurelia and the size distribution was determined with regard to growth phase and method of extraction. DNA molecules as long as 105 microns and as short as 0.2 microns were observed. It was concluded that the method of extraction affected the observed length of DNA extracted and that macronuclear DNA isolated from cells in balanced growth was less susceptible to nuclease degradation than was DNA isolated from cells in stationary phase. Renaturation studies were performed on macronuclear DNA and a kinetic complexity of 22-times E. coli DNA was determined. This value was similar to those values reported for Tetrahymena and Stylonychia macronuclear DNA. Correcting for GC base content yielded a kinetic complexity for Paramecium macronuclear DNA of 11-times E. coli DNA which corresponded to 3 X 10(10) daltons. There would be about 1400 copies of a unit genome of this complexity within each newly replicated macronucleus. Density gradient analysis indicated that the genes coding for ribosomal RNA had a greater density in CsCl than the bulk DNA. Molecular hybridization studies indicated that the genes coding for 25 S RNA represented 0.14 percent of the total macronuclear DNA. Correcting for GC base content, this corresponded to 30-35 25 S RNA genes per unit genome. These results on Paramecium are discussed in relationship to other ciliate macronuclear DNA.     

Characteristic features of the sonicated DNA of Agama agama agama L. (Reptilia,Agamidae) on hydroxyapatite columns,using mouse DNA as a reference

Hydroxyapatite column chromatography has been used to study some properties of the extensively sheared DNA of the Rainbow lizard, Agama agama agama. Reassociation studies show that the genome has a Cot_1/2 of 370. Approximately 15% of the genome is highly repetitive in nature. This repetitive fraction is resolved into thermally stable and less stable fractions. The stable fraction has a base composition of 47% GC, higher than the 40.2% GC for the native DNA. This stable fraction is believed to be of recent origin.Chromatography of the total DNA of the lizard with linear gradients of phosphate buffer containing 1 M urea resolves it into two components which were shown by thermal fractionation, also in the presence of 1 M urea, to vary in base composition. This behaviour may be characteristic of reptilian genomes and may be used as a basis for studying the structural organisation of the reptilian genome.     

Numbers of 5S and tRNA genes in macro- and micronuclei of Tetrahymena pyriformis

Macronuclei of Tetrahymena pyriformis contain approximately 200 copies of the genes for 25S and 17S ribosomal RNA (rRNA) per haploid genome. Micronuclei, however, contain only a few copies of the rRNA genes per haploid complement. Since macronuclei develop from, products of meiosis, fertilization and division of micronuclei, we suggested that the multiple copies of the rRNA genes in macronuclei are generated by amplification of the small number of genes in micronuclei (Yao et al., 1974). This process provides a simple mechanism for maintaining the homogeneity of the repeated rRNA genes. To test if amplification is a general mechanism operating on all repeated genes in Tetrahymena, we have examined the numbers of 5S RNA and tRNA genes in macro- and micronuclei. 5S RNA was purified by polyacrylamide gel electrophoresis and hybridized to saturation against macro- and micronuclear DNA. Approximately 0.013–0.014% of macronuclear DNA and about 0.009% of micronuclear DNA is complementary to 5S RNA. After correcting for the differences in the DNA sequence complexities between the two nuclei, we calculate that there are 300–350 5S genes per haploid macro- or micronuclear genome. From these data we conclude that there is little or no detectable amplification of the 5S genes in macronuclei relative to micronuclei. Similar studies using tRNA indicate that these genes are also highly repeated in both nuclei; about 800 genes are present per haploid genome. Thus, amplification from a small number of genes can be excluded as the mechanism for generating the repeated copies of the 5S and tRNA genes in Tetrahymena and it is likely that another, as yet unidentified, mechanism operates to maintain the homogeneity of these genes.     

Nuclear DNA content and base composition in 28 taxa of Musa.

The nuclear DNA content of 28 taxa of Musa was assessed by flow cytometry, using line PxPC6 of Petunia hybrida as an internal standard. The 2C DNA value of Musa balbisiana (BB genome) was 1.16 pg, whereas Musa acuminata (AA genome) had an average 2C DNA value of 1.27 pg, with a difference of 11% between its subspecies. The two haploid (IC) genomes, A and B, comprising most of the edible bananas, are therefore of similar size, 0.63 pg (610 million bp) and 0.58 pg (560 million bp), respectively. The genome of diploid Musa is thus threefold that of Arabidopsis thaliana. The genome sizes in a set of triploid Musa cultivars or clones were quite different, with 2C DNA values ranging from 1.61 to 2.23 pg. Likewise, the genome sizes of tetraploid cultivars ranged from 1.94 to 2.37 pg (2C). Apparently, tetraploids (for instance, accession I.C.2) can have a genome size that falls within the range of triploid genome sizes, and vice versa (as in the case of accession Simili Radjah). The 2C values estimated for organs such as leaf, leaf sheath, rhizome, and flower were consistent, whereas root material gave atypical results, owing to browning. The genomic base composition of these Musa taxa had a median value of 40.8% GC (SD = 0.43%).     

A new estimate of human ribosomal gene number.

Radioactively labelled DNAs (5 X 10(6) cpm/mug) complementary to human 18 S and 28 S ribosomal RNA were synthesized using RNA-directed DNA polymerase (EC 2.7.7.7). These complementary DNAs were used to measure human ribosomal gene numbers by two independent methods, both of which indicated numbers at least four-fold lower than those previously reported. First, the kinetics of the annealing of the complementary DNAs with total human placental DNA indicated that the number of both 18-S and 28-S ribosomal genes per haploid genome is approximately 50. Second, saturation experiments in which a constant amount of DNA was annealed with increasing amounts of complementary DNA also indicated that the number of 28 S ribosomal RNA genes in human placental and spleen DNA is is about 50 per haploid genome.     

Reiteration frequency of the gene coding for the small subunit of ribulose--1,5--bisphosphate carboxylase.

The messenger RNA coding for the precursor of the small subunit of ribulose--1,5--bisphosphate (Ru-P2) carboxylase has been partially purified from pea leaves. The mRNA has a size of 11.5S, is approximately 800 nucleotides long and, on cell-free translation, directs the synthesis of a single major polypeptide of 20,000 daltons. Antiserum for the small subunit of Ru-P2 carboxylase immunoprecipitated 52% of the 35S--methionine-labeled cell-free translation products. The RNA hybridizes with pea DNA with monophasic kinetics corresponding to the presence of one, or very few, gene copies per haploid genome.     

Unparalleled GC content in the plastid DNA of Selaginella

One of the more conspicuous features of plastid DNA (ptDNA) is its low guanine and cytosine (GC) content. As of February 2009, all completely-sequenced plastid genomes have a GC content below 43% except for the ptDNA of the lycophyte Selaginella uncinata, which is 55% GC. The forces driving the S. uncinata ptDNA towards G and C are undetermined, and it is unknown if other Selaginella species have GC-biased plastid genomes. This study presents the complete ptDNA sequence of Selaginella moellendorffii and compares it with the previously reported S. uncinata plastid genome. Partial ptDNA sequences from 103 different Selaginella species are also described as well as a significant proportion of the S. moellendorffii mitochondrial genome. Moreover, S. moellendorffii express sequence tags are data-mined to estimate levels of plastid and mitochondrial RNA editing. Overall, these data are used to show that: (1) there is a genus-wide GC bias in Selaginella ptDNA, which is most pronounced in South American articulate species; (2) within the Lycopsida class (and among plants in general), GC-biased ptDNA is restricted to the Selaginella genus; (3) the cause of this GC bias is arguably a combination of reduced AT-mutation pressure relative to other plastid genomes and a large number of C-to-U RNA editing sites; and (4) the mitochondrial DNA (mtDNA) of S. moellendorffii is also GC biased (even more so than the ptDNA) and is arguably the most GC-rich organelle genome observed to date—the high GC content of the mtDNA also appears to be influenced by RNA editing. Ultimately, these findings provide convincing support for the earlier proposed theory that the GC content of land-plant organelle DNA is positively correlated and directly connected to levels of organelle RNA editing.     

The isolation and characterization of a second oocyte 5s DNA from Xenopus laevis

A second and minor DNA component containing 5s RNA genes has been purified from the genomic DNA of Xenopus laevis (XIt 5S DNA). Some of its physical and chemical characteristics are described. It contains a 5S RNA gene sequence which has some oocyte and some somatic-specific residues, as well as nucleotides which differ from both types of 5S RNA. There are about 2000 of these 5S RNA genes per haploid complement of DNA compared to about 24,000 of the principal oocyte 5S RNA genes. The multiple repeating units of XIt 5S DNA are homogeneous in length (about 350 base pairs). We present evidence that XIt 5S RNA is transcribed in ovaries but not in somatic cells; XIt 5S DNA may therefore be under the same control as the major oocyte 5S DNA.     

Babesia bovis: Genome size, number of chromosomes and telomeric probe hybridisation

Pulsed field gel electrophoresis of intact chromosomes of Babesia bovis revealed four chromosomes in the haploid genome. A telomere probe, derived from Plasmodium berghei, hybridised to eight SfiI restriction fragments of genomic B. bovis DNA digests indicating the presence of four chromosomes. A small subunit (18S) ribosomal RNA gene probe hybridised to the third chromsome only. The genome size of B. bovis is estimated to be 9.4 million base pairs. The sizes of chromosomes 1, 2, 3 and 4 are estimated to be 1.4, 2.0, 2.8 and 3.2 million base pairs, respectively.     

The number and location of genes for 5S ribonucleic acid within the genome of Drosophila melanogaster

DNA was prepared from wild-type and two mutant stocks of Drosophila melanogaster that differed in their dosage of the nucleolar organizer region. The relative amounts of DNA from the nucleolar organizer region in these preparations of DNA were determined by hybridization with (3)H-labelled 28S rRNA. As expected, the amount of (3)H-labelled 28S rRNA that hybridized was directly related to the dosage of nucleolar organizer region. No positive correlation was observed between the amount of (3)H-labelled 5S RNA that hybridized and the dosage of nucleolar organizer region. Thus genes for 5S RNA are located primarily, if not exclusively, outside the nucleolar organizer region. The haploid genome of the wild-type D. melanogaster used in this work has 106 genes for 28S rRNA and 96-105 genes for 5S RNA.     

Genome complexity and organization in the red imported fire ant Solenopsis invicta Buren

The red imported fire ant Solenopsis invicta is the most destructive invading arthropod in the southern United States, yet little is known about its genome complexity and organization. Here we report the size, organization and GC content of S. invicta genome. DNA reassociation kinetics using S1 nuclease assay and a modified second-order kinetics model indicated that the S. invicta genome is approximately 0.62 picograms or 5.91 x 10(8) base pairs, composed of 36% unique, 41% moderately repetitive and 23% highly repetitive/foldback sequences. Comparison of the reassociation kinetics of short and long DNA fragments revealed that the sequence arrangement follows a pattern of short period interspersion, as in most organisms with relatively large genomes. Melting-temperature analysis showed that the GC content of the fire ant genomic DNA is 34.8%, similar to that of most eukaryotic organisms. The results reveal that the fire ant genome is much larger and more complex than those of a number of hymenopteran insects studied to date. Our study provides a foundation for further analysis and genetic manipulation of the S. invicta genome.     

Measurement of the complexity and diversity of poly(adenylic acid) containing messenger RNA from rat liver.

The complexity of rat liver poly (A)+ messenger RNA (mRNA) has been measured by analysis of the kinetics of hydridization with both complementary DNA (cDNA) and single copy DNA. The complementary DNA-poly(A)+ mRNA hybridization reaction demonstrates the existence of three abundance classes representing 18, 37, and 45% of the cDNA and 4, 290, and 24 000 different 1800-nucleotide sequences respectively. The poly(A)+ mRNA driven single copy DNA hybridization reaction reveals a single major transition accounting for 1.9% of the haploid rat genome. The kinetics of the poly(A)+ mRNA driven single copy DNA reaction suggest that approximately 45% of the mass of the mRNA population contains over 95% of the complexity. Although higher than previous estimates, the base sequence complexities of rat liver poly(A)+ mRNA measured in these two ways are in good agreement, suggesting that the technique of poly(A)+ mRNA-cDNA hybridization may be used in approximating the complexity as well as abundance of a messenger RNA population. DNA-driven cDNA reactions reveal that about 10% of rat liver poly(A)+ mRNA is transcribed from repetitive sequences in the rat genome.     

Compositional correlations in canine genome reflects similarity with human genes

The base compositional correlations that hold among various coding and noncoding regions of the canine genome have been analysed. The distribution pattern of genes, on the basis of GC(3) composition, shows a wide range similar to that observed in human. However the occurrence of maximum number of genes was observed in the range of 65-75% of GC(3) composition. The correlation between the coding DNA sequences of canine with the different noncoding regions (introns and flanking regions) is found to be significant and in many cases the degree of correlation show similarity to human genome. We found that these correlations are not limited to the GC content alone, but is holding at the level of the frequency of individual bases as well. The present study suggests that canines ideally belong to the predicted 'general mammalian pattern' of genome composition along with human beings.     

Redundant DNA of Neurospora crassa

Approximately 20% of the DNA of Neurospora crassa consists of redundant sequences. This is calculated from the reassociation rate of fragmented, denatured DNA as measured by hydroxyapatite column chromatography. The redundant DNA has a complexity of 10⁵ base pairs and a repetition frequency of up to 60 copies per genome. Its buoyant density in CsCl is 1.720 g/ml and its hypochromicity 20–24%. Base composition determination shows 54% GC content like Neurospora nuclear DNA. DNA-RNA hybridization studies indicate that rRNA and tRNA cistrons make up 2.3 and 1.2%, respectively, of the redundant fraction. Pulse-labeled RNA is shown to hybridize with both redundant and unique DNA fractions, suggesting that both fractions are transcribed.This work is supported by a grant from National Science Foundation (GB 8058) and National Institute of Health Research Career Development Award (K3GM31-238).