N-terminal Coding Sequence Research Articles

Precise quantification of translation inhibition by mRNA structures that overlap with the ribosomal footprint in N-terminal coding sequences.

A mRNA's translation rate is controlled by several sequence determinants, including the presence of RNA structures within the N-terminal regions of its coding sequences. However, the physical rules that govern when such mRNA structures will inhibit translation remain unclear. Here, we introduced systematically designed RNA hairpins into the N-terminal coding region of a reporter protein with steadily increasing distances from the start codon, followed by characterization of their mRNA and expression levels in Escherichia coli. We found that the mRNAs’ translation rates were repressed, by up to 530-fold, when mRNA structures overlapped with the ribosome's footprint. In contrast, when the mRNA structure was located outside the ribosome's footprint, translation was repressed by <2-fold. By combining our measurements with biophysical modeling, we determined that the ribosomal footprint extends 13 nucleotides into the N-terminal coding region and, when a mRNA structure overlaps or partially overlaps with the ribosomal footprint, the free energy to unfold only the overlapping structure controlled the extent of translation repression. Overall, our results provide precise quantification of the rules governing translation initiation at N-terminal coding regions, improving the predictive design of post-transcriptional regulatory elements that regulate translation rate.

Polyamines release the let-7b-mediated suppression of initiation codon recognition during the protein synthesis of EXT2.

Proteoglycans (PGs), a family of glycosaminoglycan (GAG)-protein glycoconjugates, contribute to animal physiology through interactions between their glycan chains and growth factors, chemokines and adhesion molecules. However, it remains unclear how GAG structures are changed during the aging process. Here, we found that polyamine levels are correlated with the expression level of heparan sulfate (HS) in human skin. In cultured cell lines, the EXT1 and EXT2 enzymes, initiating HS biosynthesis, were stimulated at the translational level by polyamines. Interestingly, the initiation codon recognition by 43S preinitiation complex during EXT2 translation is suppressed by let-7b, a member of the let-7 microRNA family, through binding at the N-terminal amino acid coding sequence in EXT2 mRNA. Let-7b-mediated suppression of initiation codon depends on the length of 5′-UTR of EXT2 mRNA and its suppression is inhibited in the presence of polyamines. These findings provide new insights into the HS biosynthesis related to miRNA and polyamines.

Personal reflections on RNA: an emphasis on trypanosomes.

I was drawn into the field of RNA biology as a graduate student interested in the regulation of cellular processes in pathogenic protozoa. I thought that the mechanisms that African Trypanosomes use to control the differential expression of their mitochondrial (mt) genes during their life cycle must be fundamentally important and critical to this lethal pathogen. This is because these pathogens generate energy generation by oxidative phosphorylation in the insect vector but by glycolysis in the mammalian host. My naive expectation was that this would entail conventional mechanisms such as regulation of transcription initiation to switch between the different modes of energy generation. I was surprised when it turned out to be the novel process of RNA editing, by which the coding sequences of primary mitochondrial transcripts are changed by the insertion and deletion of Us. When sequencing the unusual mt genome of T. brucei we found a frameshift in the COII gene, as did the Benne and Simpson groups in related protozoa. Benne showed that four U insertions in the mRNA eliminated the frameshift. Shortly thereafter we showed that numerous U insertions extended the N-terminal coding sequence of Cyb mRNA and created an initiation codon but this only occurred in the insect stage. Additional mRNA sequencing revealed extensive editing of COIII mRNA by the insertion and deletion, respectively, of hundreds and tens of Us. Extensive sequencing of mt mRNAs of T. brucei in my lab and of the related protozoa in other labs showed different edited mt mRNAs in these species, which is most extensive in T. brucei. Overall editing creates initiation and termination codons and creates or extends open reading frames in multiple mt mRNAs. We demonstrated translation products of the edited mRNAs by mass spectrometry showing that they are translated. Some edited mRNAs like Cyb are abundant in the insect stage, others in the mammalian stage and still others in both stages. This pattern of the differential mRNA editing matches the different energy generating processes between the life cycle stages. However, the regulatory mechanism that controls the differential editing is a mystery.

Dbf4 and Cdc7 Proteins Promote DNA Replication through Interactions with Distinct Mcm2–7 Protein Subunits

The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2-7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2-7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2-7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2ΔDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4ΔDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4ΔDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.

Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae.

Multiple dextranases from the yeast Lipomyces starkeyi

The soil yeast Lipomyces starkeyi (NCYC 1436) secretes dextranase activity into the growth medium. Resolution of a dextranase-active protein fraction by SDS-PAGE produced three protein bands, of 66 kDa, 68 kDa and 78 kDa, and isoelectric focusing of the same fraction resulted in seven protein bands, of pIs 3.50, 3.85, 4.20, 4.80, 4.85, 5.00 and 5.30. Dextranase activity was demonstrated for all the isoelectric forms, and for the 78 kDa species in the presence of SDS. Amino acid compositions of the 66 kDa, 68 kDa and 78 kDa protein bands were determined, and the N-termini of the 66 kDa and 78 kDa protein bands were sequenced: the first two amino acids at the N-terminus of each protein were alanine and valine, respectively; an alanine-valine pair is seen early in the N-terminal coding sequences of the dextranases and the isopullulanase produced by the phylogenetically disparate organisms contributing to glycosyl hydrolase family 49.

Characterization of an Osteoblast-specific Enhancer Element in the CBFA1 Gene

Cbfa1 is a critical regulator of cell differentiation expressed only in the osteochondrogenic lineage. To define the molecular basis of this cell-specific expression we analyzed the murine Cbfa1 promoter. Here we show that the first 976 bp of this promoter are specifically active in osteoblastic cells. Within this region DNase I footprinting delineated a 40-bp area (CE1) protected differently by nuclear extracts from osteoblastic cells and from non-osteoblastic cells. When multimerized, CE1 conferred an osteoblast-specific activity to a heterologous promoter in DNA transfection experiments; this enhancing ability was conserved between mouse, rat, and human CE1 present in the respective Cbfa1 promoters. CE1 site-specific mutagenesis determined that it binds NF1- and AP1-like activities. Further analyses revealed that the NF1 site acts as a repressor in non-osteoblastic cells due to the binding of NF1-A, a NF1 isoform not expressed in osteoblastic cells. In contrast, the AP1 site mediates an osteoblast-specific activation caused by the preferential binding of FosB to CE1 in osteoblastic cells. In summary, this study identified an osteoblast-specific enhancer in the Cbfa1 promoter whose activity is achieved by the combination of an inhibitory and an activatory mechanism.

Characterization of N-myristoyltransferase from Plasmodium falciparum

The gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) has been cloned from the malaria parasite Plasmodium falciparum. The gene appears to be single copy and mRNA is expressed in asexual blood-stage forms. Comparison of cDNA and genomic sequences identified three small introns. The open reading frame codes for a 410-amino-acid protein and no evidence of forms with an extended N-terminal coding sequence was obtained. Residues important in substrate binding and in the catalytic mechanism in other species are conserved. The protein was expressed from a plasmid in Escherichia coli, partially purified and shown to have enzymic activity using a synthetic peptide substrate. Comparison of the malaria parasite protein with that derived from the human gene showed a different pattern of inhibition by chemical modification. Human NMT activity was inhibited by diethylpyrocarbonate and partially inhibited by iodacetamide, whereas P. falciparum NMT activity was not inhibited by either pre-treatment. Since the enzyme in infectious fungi is a target for potential chemotherapeutic drugs, it should also be investigated in the context of parasitic infections such as that responsible for malaria.

Characterization of N-myristoyltransferase from Plasmodium falciparum

The gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) has been cloned from the malaria parasite Plasmodium falciparum. The gene appears to be single copy and mRNA is expressed in asexual blood-stage forms. Comparison of cDNA and genomic sequences identified three small introns. The open reading frame codes for a 410-amino-acid protein and no evidence of forms with an extended N-terminal coding sequence was obtained. Residues important in substrate binding and in the catalytic mechanism in other species are conserved. The protein was expressed from a plasmid in Escherichia coli, partially purified and shown to have enzymic activity using a synthetic peptide substrate. Comparison of the malaria parasite protein with that derived from the human gene showed a different pattern of inhibition by chemical modification. Human NMT activity was inhibited by diethylpyrocarbonate and partially inhibited by iodacetamide, whereas P. falciparum NMT activity was not inhibited by either pre-treatment. Since the enzyme in infectious fungi is a target for potential chemotherapeutic drugs, it should also be investigated in the context of parasitic infections such as that responsible for malaria.

The Electrogenic Sodium Bicarbonate Cotransporter: Developmental Expression in Rat Brain and Possible Role in Acid Vulnerability

The electrogenic sodium bicarbonate cotransporter (NBC) is expressed in glial cells in the brain and plays an important role in the regulation of both intracellular and extracellular pH. Differential vulnerability to acidosis between neurons and glia has been noted and may contribute to infarction after cerebral ischemia. Ionic substitution studies and inhibition of injury by 4, 4'-di-isothiocyanostilbene-2,2'-disulfonic acid suggest that NBC is involved in astrocyte vulnerability to acidic injury. Recently two NBC cDNAs differing in 5'-untranslated and N-terminal coding sequence have been cloned from kidney and pancreas. We cloned one of these cDNAs from rat brain and demonstrate here that the clone is functional by expression in Xenopus oocytes. We determined the developmental and regional expression of NBC in the brain by in situ hybridization. Expression was observed in the spinal cord at embryonic day 17, whereas expression in brain was first seen at approximately postnatal day 0 (P0), increased at P15, and persisted in the adult brain. Expression was widespread throughout the cerebellum, cortex, olfactory bulb, and subcortical structures. Cellular resolution of the in situ hybridization signal and double labeling for glial fibrillary acidic protein were consistent with a glial localization for NBC. Expression of NBC in 3T3 cells that do not normally express this transporter rendered them vulnerable to acid injury. The expression profile suggests that this transporter is critical during the later stages of brain development and could be one of the factors contributing to the different patterns of injury seen in perinatal versus adult cerebral ischemia.

Three N-terminal Variants of the AE2 Cl-/HCO3- Exchanger Are Encoded by mRNAs Transcribed from Alternative Promoters

Multiple AE2 Cl-/HCO3- exchanger mRNAs have been identified in rat. To determine the genetic basis for these mRNAs and whether they encode different variants of the exchanger, we used both rapid amplification of cDNA ends and S1 nuclease protection protocols and examined the organization of the gene. mRNAs encoding three N-terminal variants of AE2 (AE2a, AE2b, and AE2c) were identified and shown to be transcribed from alternative promoters. The AE2a transcription unit consists of 23 exons, with exons 1 and 2 containing 5'-untranslated sequence and the first 17 codons. The first exon of AE2b is located in intron 2; it contains 5'-untranslated sequence and an alternative 3-amino acid N-terminal coding sequence and is spliced to exon 3. The first exon of AE2c is located in intron 5; it consists of 5'-untranslated sequence and is spliced to exon 6, which contains the translation initiation codon corresponding to Met-200 of AE2a. Northern analysis shows that AE2a is expressed in all tissues, AE2b exhibits a more restricted distribution with highest levels in stomach, and AE2c is expressed only in stomach. Thus, the use of alternative promoters leads to the production of three N-terminal variants of AE2 that exhibit tissue-specific patterns of expression.

The human β2-adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties

We have developed a rapid and efficient expression system to study the human β 2 adrenergic receptor (hu β 2AR) in the fission yeast Schizosaccharomyces pombe. This was achieved by cloning the hu β 2AR gene, modified by replacement of the 5′ untranslated and a small part of the N-terminal coding sequence (first 14 amino acids) with the corresponding region of the yeast Saccharomyces cerevisiae STE2 (α-factor receptor) gene. The gene was then placed under the control of a S. pombe constitutive promoter for alcohol dehydrogenase (adh). Hu β 2AR expression was assessed by immunoblot analysis of the chimeric protein with an anti-STE2 serum raised against a dodecapeptide homologous to the N-terminal amino acids of STE2 and ligand binding was assayed using [ 125I]cyanopindolol. We demonstrate here that the chimeric receptor expressed in S. pombe exhibits the same characteristic ligand specificity and affinity as that of the authentic hu β 2AR. This system constitutes a convenient alternative to existing methods for studying seven transmembrane domain receptors due to its simplicity and high reproducibility.

Molecular basis for a geographic variation of varicella-zoster virus recognized by a peptide antibody.

A live attenuated varicella vaccine, derived from a Japanese isolate, is currently being widely used to modulate disease caused by varicella-zoster virus. Differentiation of the vaccine from wild-type isolates has been and will continue to be critical in the assessment of the vaccine in the United States. This has largely relied upon identification of characteristic DNA polymorphisms in the vaccine strain in the United States. This has largely relied upon identification of characteristic DNA polymorphisms in the vaccine strain. In this report, we describe the identification of a new sequence polymorphism, located in the N-terminal coding sequence of open reading frame (ORF) 10. This variation results in the synthesis of an ORF 10 protein that is differentially recognized by antibodies to an ORF 10 synthetic peptide. The variation appears to be completely restricted to Japanese strains, including that used for the live attenuated varicella vaccine. As such, this polymorphism and the antibodies that differentially recognize it could prove highly useful in the assessment of the Japanese vaccine in the United States.

Evidence for an RNA Binding Region in the Escherichia coli processing Endoribonuclease RNase E

The processing endoribonuclease RNase E (Rne), which is encoded by the rne gene, is involved in the maturation process of messenger RNAs and a ribosomal RNA. A number of deletions were constructed in order to assess functional domains of the rne gene product. The expression of the deletion constructs using a T7 promoter/RNA polymerase overproduction system led to the synthesis of truncated Rne polypeptides. The smallest gene fragment in this collection that was able to complement a temperature sensitive rnets mutation and to restore the processing of 9 S RNA was a 2.3-kilobase pair fragment with a 1.9-kilobase pair N-terminal coding sequence that mediated synthesis of a 70.8-kDa polypeptide. Antibodies raised against a truncated 110-kDa polypeptide cross-reacted with the intact rne gene product and with all of the shorter C-terminal truncated polypeptides, indicating that the N-terminal part of the molecule contained strong antigenic determinants. Furthermore, by analyzing the Rne protein and the truncated polypeptides for their ability to bind substrate RNAs, we were able to demonstrate that the central part of the Rne molecule encodes an RNA binding region. Binding to substrate RNAs correlated with the endonucleolytic activity. RNAs that are not substrates for RNase E did not bind to the protein. The two mutated Rne polypeptides expressed from the cloned gene containing either the rne-3071 or ams1 mutation also had the ability to bind 9 S RNA, while their enzymatic function was completely abolished. The data presented here suggest that the endonucleolytic activity is encoded by the N-terminal part of the Rne protein molecule and that the central part of it possesses RNA binding activity.

CRF2 alpha and CRF2 beta receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues.

We have recently described the cloning and characterization of a novel corticotropin-releasing factor receptor subtype (CRF2) from rat brain that exists in two alternatively spliced forms, CRF2 alpha and CRF2 beta. These forms differ in their N-terminal coding sequence which results in the production of two distinct receptors of 411 and 431 amino acids, respectively. To assess whether these two forms might represent distinct targets for CRF action, RNase protection and in situ hybridization studies were performed using specific N-terminal cRNA probes. The results showed a differential distribution of the mRNAs for these two receptor forms in the rat. The mRNA for CRF2 alpha is found almost exclusively in the brain, particularly in the hypothalamus, lateral septum, and olfactory bulb, whereas the mRNA for CRF2 beta appears to be both in the brain and in the periphery, with the greatest abundance in the heart and skeletal muscle. Thus, the data suggest that these alternatively spliced forms of the CRF2 receptor may represent functionally distinct CRF receptors. In addition, it highlights the importance of probe specificity for in situ hybridization studies.

Modification of a xylanase cDNA isolated from an anaerobic fungus Neocallimastix patriciarum for high-level expression in Escherichia coli

A Neocallimastix patriciarum xylanase cDNA with the core coding sequence essentially identical to xynA was isolated and modified for high-level expression in Escherichia coli. The xylanase cDNA was truncated into individual catalytic domains, which were modified at the N-terminus. These modified xylanases were synthesised as non-fusion proteins under the control of the tac promoter. High-level expression was obtained with the modified domain II construct, accounting for approx. 25% of total cellular protein. However, with the same vector and expression cassette, expression levels of constructs containing domain I or domains I and II fused in tandem were very low. RNA analysis revealed that the striking difference in expression levels of these three constructs was not due to transcription efficiency, but was mainly related to transcript stability. Further analysis of the domain II construct revealed that the high-level expression of the domain II xylanase was largely attributed to the presence of a favourable N-terminal coding sequence, as mutation at the N-terminus of the domain II dramatically reduced the expression level. The modified domain II xylanase produced in E. coli had a specific activity of 1229 U mg −1 protein at pH 7 and 50 °C without purification. The availability of a recombinant fungal xylanase with high specific activity and in high yield offers a potentially attractive source of xylanase for industrial applications.

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5' terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5'-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.

Alpha 2-chimerin, an SH2-containing GTPase-activating protein for the ras-related protein p21rac derived by alternate splicing of the human n-chimerin gene, is selectively expressed in brain regions and testes.

n-Chimerin (alpha 1-chimerin) is a brain GTPase-activating protein (GAP) for the ras-related p21rac. We now report the occurrence of another form of chimerin, termed alpha 2-chimerin. This is the product of an alternately spliced transcript of the human n-chimerin gene encoding an N-terminal SH2 (src homology 2) domain in addition to the phorbol ester receptor and GAP domains. alpha 1- and alpha 2-chimerin mRNAs were expressed differently. In the rat brain, only alpha 1-chimerin mRNA was expressed in cerebellar Purkinje cells, although both alpha 1- and alpha 2-chimerin mRNAs occurred in neurons in the cerebral cortex, hippocampus, and thalamus. Only alpha 2-chimerin RNA was expressed in rat testes, in early pachytene spermatocytes. A 45-kDa SH2-containing chimerin corresponding to the alpha 2 form was purified from rat brain. As with Escherichia coli 45-kDa recombinant alpha 2-chimerin, purified brain alpha 2-chimerin exhibited racGAP activity which was stimulated by phosphatidylserine. The recombinant SH2 domain bound several 32P-labelled phosphoproteins of PC12 cells, whose phosphorylation increased in response to trophic factors, including nerve growth factor. To examine the relationships of alpha 1- and alpha 2-chimerin transcripts, human genomic DNA clones were characterized. In alpha 2-chimerin mRNA, a 3' splice acceptor site within exon 1 of alpha 1-chimerin mRNA was used, replacing its 5' untranslated region and N-terminal coding sequence. The single human n-chimerin gene was mapped to chromosome 2q31-q32.1, colocalizing with the CRE-BP1 transcription factor gene (2q32). It contained several splice junctions conserved with the sequence-related protein kinase C and bcr genes. alpha 2-Chimerin is only the second SH2-containing GAP and the first example of an SH2 domain generated by alternate splicing.

The VirD2 protein of Agrobacterium tumefaciens carries nuclear localization signals important for transfer of T-DNA to plant.

Agrobacterium tumefaciens is able to transfer a piece of DNA, the T-DNA, to the nucleus of the plant cell. The VirD2 protein is required for the production of the T-DNA, it is tightly linked to the T-DNA and it is thought to direct it to the plant genome. Two nuclear localization signals (NLS), one in the N-terminal part and one in the C-terminal part of the VirD2 protein, have been shown to be able to target marker proteins to the plant nucleus. Here we analyze nuclear entry of the T-DNA complex using a new and very sensitive assay for T-DNA transfer. We show that optimal T-DNA transfer requires the VirD2 NLS located in the C-terminal part of the protein, whereas mutations in the N-terminal NLS coding sequence seem to have no effect on T-DNA transfer.

Selection of Arabidopsis cDNAs that partially correct phenotypes of Escherichia coli DNA-damage-sensitive mutants and analysis of two plant cDNAs that appear to express UV-specific dark repair activities.

To resist terrestrial UV radiation, plants employ DNA-damage-repair/toleration (DRT) activities, as well as shielding mechanisms. Little is known about the structure and regulation of plant DRT genes. We isolated DRT cDNAs from Arabidopsis thaliana, by selecting for complementation of Escherichia coli mutants lacking all bacterial defenses against UV-light damage to DNA. These mutants are phenotypically deficient in recombinational and mutagenic toleration (RecA-), excision repair (Uvr-) and photoreactivation (Phr-). Among 840 survivors of heavily UV-irradiated (10(-7) survival) mutants harboring plasmids derived from an Arabidopsis cDNA library in the vector lambda YES, we identified four unique plant cDNAs, designated DRT100, DRT101, DRT102, and DRT103. Drt101 and Drt102 activity were specific for UV-light damage, and complemented both UvrB- and UvrC- phenotypes in the dark. Apparent Uvr- correction efficiencies were 1 to 40% for Drt101, and 0.2 to 15% for Drt102, depending on the UV fluence. Drt101 and Drt102 showed no extensive amino-acid homology with any known DNA-repair proteins. Drt100 appeared to correct RecA-, rather than Uvr-, phenotypes. Although the light dependence of Drt103 activity was consistent with its identification as a photoreactivating enzyme, its predicted amino-acid sequence did not resemble known photolyase sequences. The N-terminal coding sequence of Drt101 suggests that it is targeted to chloroplasts, as reported for Drt100. These cDNAs afforded only modest increases in survival during the original selection procedure. The fact that they were readily isolated nevertheless suggests that selections may be made powerful enough to overcome barriers to expression and function in bacteria, at least for cDNAs of reasonable abundance.