Evolutionary diversity of eukaryotic small-subunit rRNA genes.

The small-subunit rRNA gene sequences of the flagellated protists Euglena gracilis and Trypanosoma brucei were determined and compared to those of other eukaryotes. A phylogenetic tree was constructed in which the earliest branching among the eukaryotes is represented by E. gracilis. The E. gracilis divergence far antedates a period of massive evolutionary radiation that gave rise to the plants, animals, fungi, and certain groups of protists such as ciliates and the acanthamoebae. The genetic diversity in this collection of eukaryotes is seen to exceed that displayed within either the eubacterial or the archaebacterial lines of descent.     

Functional genomics reveals a family of eukaryotic oxidation protection genes

Reactive oxygen species (ROS) are toxic compounds produced by normal metabolic processes. Their reactivity with cellular components is a major stress for aerobic cells that results in lipid, protein, and DNA damage. ROS-mediated DNA damage contributes to spontaneous mutagenesis, and cells deficient in repair and protective mechanisms have elevated levels of spontaneous mutations. In Escherichia coli a large number of genes are involved in the repair of oxidative DNA damage and its prevention by detoxification of ROS. In humans, the genes required for these processes are not well defined. In this report we describe the human OXR1 (oxidation resistance) gene discovered in a search for human genes that function in protection against oxidative damage. OXR1 is a member of a conserved family of genes found in eukaryotes but not in prokaryotes. We also outline the procedures developed to identify human genes involved in the prevention and repair of oxidative damage that were used to identify the human OXR1 gene. This procedure makes use of the spontaneous mutator phenotype of E. coli oxidative repair-deficient mutants and identifies genes of interest by screening for antimutator activity resulting from cDNA expression.     

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.     

Cotransfer of linked eukaryotic genes and efficient transfer of hypoxanthine phosphoribosyltransferase by DNA-mediated gene transfer.

The efficiency of DNA-mediated transfer of the gene (hprt) for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is dependent upon the recipient cell used. hprt has been transferred into mouse TG8 or Chinese hamster CHTG49 cells at a high frequency, similar to the frequency of the gene (tk) for thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) transfer into mouse LMTK- cells (i.e., 10(-6)). In contrast, the frequency of transfer of hprt into mouse A9 cells was about two orders of magnitude less. The identification of efficient recipient cells for hprt transfer permits the use of DNA-mediated transfer as a bioassay for the gene. Cotransfer of the linked tk gene and the gene (galk) for galactokinase (ATP: D-galactose 1-phosphotransferase, EC 2.7.1.6) to LMTK- cells has been detected once among 87 tk transferrents. This suggests that the distance between the tk and galk genes in the Chinese hamster genome may be smaller than was previously thought. Significant differences between chromosome-mediated and DNA-mediated gene transfer were observed with respect to both the size of the transferred functional genetic fragment and the recipient cell specificity.     

Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles

We have employed microprojectiles to deliver genes involved in anthocyanin biosynthesis to cells within intact aleurone and embryo tissues of maize. Clones of the A1 or Bz1 genes were introduced into aleurone tissue that lacked anthocyanins due to mutations of the endogenous A1 or Bz1 gene. Following bombardment, cells within the aleurone developed purple pigmentation, indicating that the mutation in the a1 or bz1 genotypes was corrected by the introduced gene. To analyze the expression of these genes in different genetic backgrounds, chimeric genes containing the 5′ and 3′ regions of the A1 or Bz1 genes fused to a luciferase coding region were constructed. These constructs were introduced into aleurones of genotypes carrying either dominant or recessive alleles of the C1 and R genes, which are known to regulate anthocyanin production. Levels of luciferase activity in permissive backgrounds (C1, R) were 30- to 200-fold greater than those detected in tissue carrying one or both of the recessive alleles (c1, r) of these genes. These results show that genes delivered to intact tissues by microprojectiles are regulated in a manner similar to the endogenous genes. The transfer of genes directly to intact tissues provides a rapid means for analyzing the genetic and tissue-specific regulation of gene expression.     

Regulation of expression of somatostatin genes by sex steroid hormones in goldfish forebrain

Recently, our laboratory has identified three distinct pre-pro-somatostatin (PSS) genes in goldfish brain: PSS-I encodes for somatostatin (SRIH)-14, PSS-II encodes SRIH-28, which contains [Glu(1), Tyr(7), Gly(10)] SRIH-14 at its C-terminus, and PSS-III encodes [Pro(2)] SRIH-14. In goldfish, increasing levels of the sex steroid estradiol increase the plasma levels of growth hormone (GH). However, whether sex steroids act at the level of the brain to regulate GH release is unclear. In the present study, the effects of sex steroids on the expression of the three PSS genes in goldfish forebrain were examined. The results demonstrate that treatment with estradiol significantly increases the expression of PSS-I and PSS-III genes in both male and female fish. The effects of estradiol were evident after only 2.5 days of treatment. Testosterone treatment increased the expression of PSS-I and PSS-III genes in female but not male fish, and only at the highest dose used. In addition, the effects of testosterone were evident only after treatment for 5 or 10 days and were blocked by an aromatase inhibitor, suggesting that testosterone must be converted to estradiol to exhibit the effect. Neither estradiol nor testosterone treatment had effects on the expression of the PSS-II gene. These results suggest that sex steroids can act either directly or indirectly on the brain to regulate PSS-I and PSS-III gene expression, influencing in turn the regulation of GH secretion.     

The organization of Physcomitrella patensRAD51 genes is unique among eukaryotic organisms

Genetic recombination pathways and genes are well studied, but relatively little is known in plants, especially in lower plants. To study the recombination apparatus of a lower land plant, a recombination gene well characterized particularly in yeast, mouse, and man, the RAD51 gene, was isolated from the moss Physcomitrella patens and characterized. Two highly homologous RAD51 genes were found to be present. Duplicated RAD51 genes have been found thus far exclusively in eukaryotes with duplicated genomes. Therefore the presence of two highly homologous genes suggests a recent genome duplication event in the ancestry of Physcomitrella. Comparison of the protein sequences to Rad51 proteins from other organisms showed that both RAD51 genes originated within the group of plant Rad51 proteins. However, the two proteins form a separate clade in a phylogenetic tree of plant Rad51 proteins. In contrast to RAD51 genes from other multicellular eukaryotes, the Physcomitrella genes are not interrupted by introns. Because introns are a common feature of Physcomitrella genes, the lack of introns in the RAD51 genes is unusual and may indicate the presence of an unusual recombination apparatus in this organism. The presence of duplicated intronless RAD51 genes is unique among eukaryotes. Studies of further members of this lineage are needed to determine whether this feature may be typical of lower plants.     

Regulation of double-stranded RNA-activated eukaryotic initiation factor 2α kinase by type 2 protein phosphatase in reticulocyte lysates

Protein synthesis initiation in reticulocyte lysates is inhibited by low concentrations (1-20 ng/ml) of double-stranded RNA (ds RNA) due to the activation of a ds RNA-dependent cAMP-independent protein kinase (ds I) that phosphorylates the α subunit of the eukaryotic initiation factor eIF-2. In lysates, ds I is present in the latent inactive form and is associated with the ribosome complement. Latent ds I is solubilized by extraction with high-salt buffers and can be purified in its latent form. Activation of purified latent ds I requires ds RNA and ATP and is accompanied by the ds RNA-dependent autophosphorylation of a polypeptide doublet of 70,000 and 72,000 daltons (“70k/72k”), which represent different phosphorylated states of the same polypeptide. These are phosphorylated in the sequence 70k→72k; increased phosphorylation of 72k is associated with increased ds I activation. Lysates (or Sepharose 6B ribosomes) treated with ds RNA display a similar ds I phosphoprotein profile, and this is accompanied by the phosphorylation of endogenous eIF-2α (38,000 daltons). Delayed ³²P pulses in ds RNA-inhibited lysates indicate that the phosphates on ds I and eIF-2α turn over. Under defined conditions, activated ds I in lysates is selectively dephosphorylated by endogenous protein phosphatase(s), and this is accompanied by the dephosphorylation of eIF-2α. Similarly, purified activated ds I is rapidly dephosphorylated by unfractionated lysate protein phosphatase(s) and by type 2 protein phosphatase but not by type 1 protein phosphatase. The dephosphorylation of ds I occurs in the sequence 72k→70k and is correlated with ds I inactivation. The heat-stable protein phosphatase inhibitor-2, which selectively blocks type 1 protein phosphatase, does not significantly affect the dephosphorylation of ds I by type 2 protein phosphatase or by unfractionated lysate phosphatases. The data support the conclusion that a ds I phosphatase activity with type 2 characteristics is involved in the regulation of ds I activity.     

Possible evolution of splice-junction signals in eukaryotic genes from stop codons.

Splice-junction sequence signals are strongly conserved structural components of eukaryotic genes. These sequences border exon/intron junctions and aid in the process of removing introns by the RNA splicing machinery. Although substantial research has been undertaken to understand the mechanism of splicing, little is known about the origin and evolution of these splice signal sequences. Based on the previously published theory that the primitive genes evolved in pieces from primordial genetic sequences to avoid the interfering stop codons, a "stop-codon walk" mechanism is proposed in this paper to have assisted in the evolution of coding genes. This mechanism predicts the presence of stop codons in splice-junction signals inside the introns. Evidence of the consistent presence of stop codons in the splice-junction signals, in a position where they are expected, is shown by the analysis of codon statistics in these signal sequences in the GenBank databank. The results suggest that the splice-junction signals may have evolved from stop codons as a consequence of a selective pressure to avoid stop codons during the original evolution of coding genes. They also suggest that other splice signals within the introns, such as the branch-point sequence, may have evolved from stop codons for similar reasons.     

Regulation of protein synthesis by phosphorylation of eukaryotic initiation factor 2 alpha in intact reticulocytes and reticulocyte lysates.

Studies in intact rabbit reticulocytes and reticulocyte lysates provide further evidence of a functional role for the phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) in the regulation of initiation of protein synthesis in eukaryotic cells. In intact reticulocytes treated with isonicotinic acid hydrazide to inhibit heme synthesis, the phosphorylation of eIF-2 alpha was significantly greater than in control cells. In heme-deficient reticulocyte lysates and in lysates treated with double-stranded RNA, significant phosphorylation of eIF-2 alpha occurred prior to the onset of inhibition of protein synthesis; a large proportion, however, of the total eIF-2 alpha remained unphosphorylated. These findings indicate that a modest concentration of phosphorylated eIF-2 alpha can suffice to inhibit initiation, and they suggest that one of the factors with which eIF-2 must interact may be rate limiting, especially when eIF-2 alpha is phosphorylated.