It was previously shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, although the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the alanyl-tRNA synthetase gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments. It was previously shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, although the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the alanyl-tRNA synthetase gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments. Typically there are 20 aminoacyl-tRNA synthetases in prokaryotes, one for each amino acid (1Carter Jr., C.W. Annu. Rev. Biochem. 1993; 62: 715-748Crossref PubMed Scopus (327) Google Scholar, 2Martinis S.A. Schimmel P. Neidhardt F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C1996: 887-901Google Scholar, 3Giege´ R. Sissler M. Florentz C. Nucleic Acids Res. 1998; 26: 5017-5035Crossref PubMed Scopus (633) Google Scholar, 4Pelchat M. Lapointe J. Biochem. Cell Biol. 1999; 77: 343-347Crossref PubMed Google Scholar). These enzymes each catalyze the formation of an aminoacyl-tRNA by attaching a particular amino acid to the 3′-end of its cognate tRNA, with accompanying hydrolysis of ATP to AMP and pyrophosphate. The activated amino acid, i.e. aminoacyl-tRNA, is then transferred to ribosome for protein synthesis. In eukaryotes protein synthesis occurs not only in the cytoplasm but also in organelles, such as mitochondria and chloroplasts (5Mare´chal-Drouard L. Weil J.H. Dietrich A. Annu. Rev. Cell Biol. 1993; 8: 115-131Google Scholar). Compartmentalization of the protein synthesis machinery within the cytoplasm and organelles of eukaryotes leads to isoaccepting tRNA species that are distinguished by nucleotide sequence, subcellular location, and enzyme specificity. Thus, eukaryotes such as yeast commonly have two genes that encode distinct sets of proteins for each aminoacylation activity, one localized to the cytoplasm and the other to the mitochondria. Each set aminoacylates the isoaccepting tRNAs within its respective cell compartment. Except for some algae (6Steinmetz A. Weil J.H. Methods Enzymol. 1986; 118: 212-231Crossref Scopus (28) Google Scholar), all aminoacyl-tRNA synthetases are encoded by nuclear genes regardless of the cell compartments to which they are confined. In contrast to most known eukaryotic tRNA synthetases, two Saccharomyces cerevisiae genes, HTS1 (the gene encoding his-tidyl-tRNA synthetase) (7Natsoulis G. Hilger F. Fink G.R. Cell. 1986; 46: 235-243Abstract Full Text PDF PubMed Scopus (196) Google Scholar) and VAS1 (the gene encoding valyl-tRNA synthetase (ValRS)) (8Chatton B. Walter P. Ebel J.-P. Lacroute F. Fasiolo F. J. Biol. Chem. 1988; 263: 52-57Abstract Full Text PDF PubMed Google Scholar), specify both the mitochondrial and cytosolic forms through alternative initiation from two inframe AUG codons. Each of these genes encodes mRNAs with distinct 5′-ends. Some of these mRNAs have their 5′-ends located upstream of the first AUG codon, whereas others have their 5′-ends located between the first and second AUG codons. The mitochondrial form of the enzyme is translated from the first AUG on the “long” messages, whereas the cytosolic form is translated from the second AUG on the “short” messages. As a consequence, the mitochondrial enzymes have the same polypeptide sequences as their cytosolic counterparts, except for a short amino-terminal mitochondrial targeting sequence. The transit peptide is subsequently cleaved away upon import into the mitochondria. Because the two isoforms are targeted to different subcellular compartments, they cannot substitute for each other in vivo (7Natsoulis G. Hilger F. Fink G.R. Cell. 1986; 46: 235-243Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 8Chatton B. Walter P. Ebel J.-P. Lacroute F. Fasiolo F. J. Biol. Chem. 1988; 263: 52-57Abstract Full Text PDF PubMed Google Scholar). A similar scenario has been observed for the genes that encode the mitochondrial and cytoplasmic forms of Arabidopsis thaliana alanyl-tRNA synthetase (AlaRS), 3The abbreviations used are: AlaRS, alanyl-tRNA synthetase; ADH, alcohol dehydrogenase; 5-FOA, 5-fluoroorotic acid; ValRS, valyl-tRNA synthetase; YPG, yeast extract-peptone-glycerol; RACE, rapid amplification of cDNA ends; RT, reverse transcription. 3The abbreviations used are: AlaRS, alanyl-tRNA synthetase; ADH, alcohol dehydrogenase; 5-FOA, 5-fluoroorotic acid; ValRS, valyl-tRNA synthetase; YPG, yeast extract-peptone-glycerol; RACE, rapid amplification of cDNA ends; RT, reverse transcription. threonyl-tRNA synthetase, and ValRS (9Souciet G. Menand B. Ovesna J. Cosset A. Dietrich A. Wintz H. Eur. J. Biochem. 1999; 266: 848-854Crossref PubMed Scopus (55) Google Scholar). Recently it was shown that ALA1, the only gene coding for AlaRS in S. cerevisiae, also encodes distinct protein isoforms (10Tang H.L. Yeh L.S. Chen N.K. Ripmaster T. Schimmel P. Wang C.C. J. Biol. Chem. 2004; 279: 49656-49663Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Chang K.J. Lin G. Men L.C. Wang C.C. J. Biol. Chem. 2006; 281: 7775-7783Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Although the cytoplasmic form is initiated from a canonical AUG triplet, its mitochondrial counterpart is initiated from two successive in-frame ACG triplets that are located 23 codons upstream of the AUG initiator, i.e. ACG(–25)/ACG(–24). These two forms function exclusively in their respective compartments and, thus, cannot substitute for each other under normal conditions. A similar scenario has been observed in GRS1 (12Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), the only active yeast gene coding for glycyl-tRNA synthetase. Because to date examples of native non-AUG initiation are still rare in low eukaryotes (10Tang H.L. Yeh L.S. Chen N.K. Ripmaster T. Schimmel P. Wang C.C. J. Biol. Chem. 2004; 279: 49656-49663Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Chang K.J. Lin G. Men L.C. Wang C.C. J. Biol. Chem. 2006; 281: 7775-7783Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 12Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), we wondered whether a similar mechanism of translation initiation has been conserved in the AlaRS genes of other yeasts during evolution. In addition, we wondered whether the AlaRS gene of a closely related yeast species, such as Candida albicans, also provides function in both compartments and whether it can surmount the species barrier and charge the tRNAs of S. cerevisiae. It is our hope that results obtained from this study could provide further insight into the bifunctional nature of a particular nuclear gene and the diversity of mechanisms by which protein isoforms can be partitioned between two distinct compartments. In the work described here we presented experimental evidence that an ALA1 homologue of C. albicans (designated here as CaALA1) can rescue both the cytoplasmic and mitochondrial defects of a S. cerevisiae ala1– strain. Similar to the ALA1 gene in S. cerevisiae, two protein isoforms with distinct amino termini are alternatively generated from this gene; however, no non-canonical initiators are involved in this case. Instead, these isoforms are initiated from two in-frame AUG triplets. Even more unexpectedly, whereas the short form that is initiated from the second AUG is confined in the cytoplasm, the longer form that is initiated from the first AUG is dual-targeted and, thus, bifunctional. The implications of these observations will be further discussed in the context of co-evolution of tRNAs and their cognate tRNA synthetases. Construction of Plasmids—Cloning of CaALA1 from C. albicans followed standard protocols (13Wang W. Malcolm B.A. Biotechniques. 1999; 26: 680-682Crossref PubMed Scopus (490) Google Scholar). The wild-type CaALA1 sequence (base pairs –300∼+2910 relative to ATG1) was amplified by PCR and cloned into pRS315 (a low copy number yeast vector) or pRS425 (a high copy number yeast vector). A short sequence coding for a His6 tag or FLAG was subsequently inserted in-frame into the 3′-end of the CaALA1 open reading frame. Various point mutations, such as ATG1/ATA2 to TCT/AGA and ATG9 to GCG, were introduced into the wild-type clone following standard protocols (the number 1 in ATG1 refers to the codon position in the open reading frame). To clone CaALA1 in pADH (a high copy number yeast vector with an ADH promoter), a segment of CaALA1 DNA containing base pairs –40∼+2910 relative to ATG1 was amplified by PCR as an EagI/XhoI fragment and cloned into appropriate sites of this vector. Cloning of CaALA1-VAS1c constructs followed a strategy described earlier (12Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Basically, various CaALA1 sequences (–370∼+102 bp, –370∼+126 bp, or –370∼+159 bp) were PCR-amplified as EagI-SpeI fragments and fused in-frame to the EagI/SpeI sites 5′ to VAS1c (the open reading frame coding for the cytoplasmic form of ValRS) cloned in a low copy number vector pRS315, resulting in various CaALA1-VAS1c constructs in which the ATG initiator for the cytoplasmic form of ValRS had been mutated. Mapping the 5′-Ends of CaALA1 Transcripts—Identification of the 5′-ends of CaALA1 transcripts was carried out with 5′-RACE (rapid amplification of cDNA ends; Invitrogen). Briefly, total RNA isolated from C. albicans was first treated with alkaline phosphatase to remove the 5′-phosphate group from truncated mRNA and non-mRNA and then with tobacco acid pyrophosphatase to remove the 5′-cap from intact full-length mRNA. An RNA oligonucleotide was subsequently fused to the 5′-end of the decapped mRNA with RNA ligase. The 5′-end modified mRNA was transcribed with SuperScript III reverse transcriptase into first strand cDNAs using an “antisense” CaALA1-specific primer that was annealed to a region 630-bp downstream of ATG1. The reaction mixture was treated with RNase H, and the first strand cDNA products were then amplified via PCR using Pfu DNA polymerase with a primer (provided by the manufacturer) annealed to the 5′-end of the cDNA and a nested CaALA1-specific primer annealed 600 bp downstream of ATG1. After PCR-driven amplification, the double-stranded cDNA products were cloned and sequenced. Sequencing of the Mitochondrial Form of CaAlaRS—Determination of the amino terminus of the processed mitochondrial form of CaAlaRS was carried out by the Edman degradation method. First, mitochondria were isolated from transformants carrying the wild-type (pIVY97) and ATG9 mutant (pIVY118) constructs (14Daum G. Bohni P.C. Schatz G. J. Biol. Chem. 1982; 257: 13028-13033Abstract Full Text PDF PubMed Google Scholar), and the His6-tagged proteins expressed were purified by nickel-nitrilotriacetic acid column chromatography. After SDS-polyacrylamide gel electrophoresis, the proteins were transferred to a nitrocellulose membrane and stained with Amido Black, and the protein band of the correct size was removed and sequenced. Complementation Assays for the Cytoplasmic Function of ALA1—The yeast ALA1 knock-out strain TRY11 was as described (15Ripmaster T.L. Shiba K. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4932-4936Crossref PubMed Scopus (40) Google Scholar). This strain is maintained by a plasmid encoding AlaRS and the URA3 marker. Complementation assays for the cytoplasmic function of plasmid-borne ALA1 and derivatives were carried out by introducing a test plasmid into TRY11 and determining the ability of transformants to grow in the presence of 5-fluoroorotic acid (5-FOA). The cultures were incubated at 30 °C for 3–5 days or until colonies appeared (photos for the complementation assays were taken at day 3 after incubation). The transformants evicted the maintenance plasmid with the URA3 marker in the presence of 5-FOA. Thus, only an enzyme with the cytoplasmic AlaRS activity encoded by the second plasmid (with the LEU2 marker) could rescue the growth defect. Complementation Assays for the Mitochondrial Function of ALA1—Complementation assays for the mitochondrial function of plasmid-borne ALA1 and derivatives were carried out by introducing a test plasmid (carrying a LEU2 marker) and a second maintenance plasmid (carrying a HIS3 marker) into TRY11 and selecting on a plate containing 5-FOA. The second maintenance plasmid used in this assay contained ALA1(I(–1)stop), which expresses a functional cytoplasmic AlaRS but is defective in mitochondrial AlaRS activity. In the presence of 5-FOA, the first maintenance plasmid (containing a URA3 marker) was evicted from the co-transformants, whereas the second maintenance plasmid was retained. Thus, all co-transformants survived 5-FOA selections due to the presence of the cytoplasmic AlaRS derived from the second maintenance plasmid. The co-transformants were further tested on YPG plates for their mitochondrial phenotypes at 30 °C, with results documented at day 3 after plating. Because a yeast cell cannot survive on glycerol without functional mitochondria, the co-transformants do not grow on YPG plates unless a functional mitochondrial AlaRS is present. Complementation assays for various VAS1 constructs and their derivatives were conducted essentially the same way as those for ALA1 constructs, except that a VAS1 knock-out strain, CW1, was used as the test strain (16Wang C.C. Chang K.J. Tang H.L. Hsieh C.J. Schimmel P. Biochemistry. 2003; 42: 1646-1651Crossref PubMed Scopus (25) Google Scholar). Western Blot—The protein expression patterns of various CaALA1 constructs were determined by a chemiluminescence-based Western blot analysis following standard protocols. The CaALA1 constructs were first transformed into INVSc1 (Novagen), and the resultant transformants were subsequently grown in a selection medium lacking leucine. The total, cytoplasmic, and mitochondrial fractions were prepared from each of the transformants according to the protocols described by Daum et al. (14Daum G. Bohni P.C. Schatz G. J. Biol. Chem. 1982; 257: 13028-13033Abstract Full Text PDF PubMed Google Scholar). 45 μg of the protein extracts were loaded onto a gel (size, 8 × 10 cm) containing 8% polyacrylamide and electrophoresed at 130 volts for 1∼2 h. The resolved proteins were transferred onto a nitrocellulose membrane using a semidry blotting device. The membrane was hybridized with a horseradish peroxidase-conjugated anti-His6 tag (Invitrogen) or anti-FLAG antibody (Sigma) and then exposed to x-ray film after the addition of the appropriate substrates. Growth Curve Assay—Growth curve assays for the plasmid-borne mitochondrial AlaRS activities were carried out in YPG broth. TRY11 was first co-transformed with a second maintenance plasmid (carrying a HIS3 marker) and a test plasmid (carrying a LEU2 marker), and the resultant co-transformants were plated on a FOA plate. After FOA selections, one colony of the survivors was picked and inoculated into 3 ml of SD broth lacking histidine and leucine and grown to stationary phase. The cells were washed three times with YPG broth, and appropriate amounts were transferred to a flask containing 10 ml of YPG broth to a final cell density of A600 = 0.1. The cell culture was shaken in a 30 °C incubator, and the cell density of the culture was checked every 4 h for a period of 48 h. Cross-species Complementation of an S. cerevisiae ala1– Strain by a Homologue of C. albicans—Unlike most yeast tRNA synthetases that have two distinct nuclear genes (one coding for the cytoplasmic enzyme and the other for its mitochondrial counterpart), CaALA1 appears to be the only ALA1 homologue in the yeast C. albicans. We wondered whether this gene actually provides AlaRS function in vivo and whether it could code for both cytoplasmic and mitochondrial activities, as with the case of ALA1 in S. cerevisiae. To further our understanding on this gene, we first scanned the 5′-terminal nucleotide sequences of this gene for potential translation start codons that might be involved in the synthesis of protein isoforms. As shown in Fig. 1A, there are two nearby in-frame ATG codons, i.e. ATG1 and ATG9, close to the 5′-end of its open reading frame. In addition, four potential non-ATG initiators, i.e. non-ATG codons that differ from ATG by a single nucleotide, are present in the sequence between ATG1 and TGA(-47), the closest termination codon. We wondered which of these triplets is the authentic start sites for CaALA1. Before proceeding, the transcription profiles of this gene in vivo were elucidated using 5′-RACE. Fig. 1B showed that a single transcript, with its 5′-end mapped to nucleotide position –24 relative to ATG1, was amplified by RT-PCR using total RNA extracts of C. albicans as the templates (Fig. 1B). This transcript was, thus, considered to be the template for protein translation. In addition, sequences of the cDNAs (∼600 bp determined) obtained from RT-PCR were identical to those of the genomic DNA (data not shown), suggesting that the possibilities of alternative splicing at the 5′-end of CaALA1 mRNAs and translation initiation from an AUG codon spliced from afar could be ruled out. Comparison of the protein sequences among AlaRSs of different origins showed that CaAlaRS (deduced from its putative open reading frame starting at ATG1) shares a significantly higher sequence identity to those from S. cerevisiae (cytoplasmic form) (∼68%) and Schizosaccharomyces pombe (∼61%) than to the Escherichia coli enzyme (∼39%) (Fig. 1C). Most intriguingly, this protein appears to have an amino-terminal 15-residue appendage that is absent from the other yeast cytoplasmic enzymes compared (Fig. 1D). It should be noted that the cytoplasmic form of AlaRS of S. cerevisiae starts from the second Met on the sequence shown in Fig. 1D. We surmised that if this protein does have mitochondrial function, this appendage could serve as at least part of its mitochondrial targeting signal. Because the genetic system has not been well developed for C. albicans, we wondered whether we could test the biological functions of CaALA1 in a closely related and well developed yeast system such as S. cerevisiae. To this end, CaALA1 cloned in various vectors was transformed into a S. cerevisiae ala1– yeast strain, TRY11, and tested for its complementing activity. As shown in Fig. 2, the wild-type CaALA1 gene cloned in pRS315 (a low copy number vector), pRS425 (a high copy number vector), or pADH (a high copy number vector with a constitutive ADH promoter) efficiently rescued both the cytoplasmic and mitochondrial defects of TRY11, i.e. transformants carrying the plasmid-borne CaALA1 gene formed colonies after 2∼3 days of growth on FOA (Fig. 2B) and YPG (Fig. 2C) plates, respectively. These results indicated that the homologous gene from C. albicans could overcome the species barrier and encodes both cytoplasmic and mitochondrial AlaRS activities. Generation of Two Functionally Overlapping Protein Isoforms through Alternative Translation Initiation—The question arose as to how many protein isoforms are generated from CaALA1 and whether the upstream non-ATG triplets (relative to ATG1) are involved in the translation initiation. To shed light on this matter, various CaALA1 constructs were cloned using pRS425 as the vector and tested for their complementation activities. First, the codon at position –1 was mutated to a stop codon TAA to block all the possible translational events initiated upstream of ATG1 and then tested for its effect on the cytoplasmic and mitochondrial functions of this gene. Fig. 3 shows that the newly introduced stop codon affected neither the mitochondrial nor cytoplasmic function of this gene (see pIVY102), suggesting that the upstream potential non-ATG initiators are not involved in the synthesis of the alanine enzyme(s). We next aimed at the two nearby ATG triplets, i.e. ATG1 and ATG9, for their possible participation in translation. Mutation of ATG1 (see pIVY100 or pSAM35) or insertion of two nucleotides between ATG1 and ATG9 (causing ATG1 to be out-of-frame with respect to the rest of the open reading frame) (see pSAM25) specifically impaired the mitochondrial function of this gene (Fig. 3, B and C), suggesting that ATG1 was the sole initiator responsible for the translation of the mitochondrial form and the cytoplasmic form was initiated elsewhere, possibly from ATG9. Further mutation of ATG9 in pIVY100 (resulting in pIVY104) abolished the remaining cytoplasmic activity, indicating that the cytoplasmic function of pIVY100 was indeed provided by protein product initiated at ATG9. However, much to our surprise, mutation of ATG9 alone did not impair the cytoplasmic function as expected (see pSAM33); instead, the ATG9 mutant still retained both activities. These results suggested that the long form that is initiated from ATG1 provided both the cytoplasmic and mitochondrial functions, whereas the shorter form that is initiated from ATG9 provided only the cytoplasmic activity. Given that the out-of-frame mutant (see pSAM25) still retained the cytoplasmic function, it is likely that the second ATG triplet can be recognized by scanning ribosomes as a remedial translation start site even in the presence of the first ATG triplet. Therefore, the cytoplasmic function of this gene is probably contributed by both isoforms under normal conditions. It is noteworthy that mutation of ATG1 to GCG (resulting in pSAM35) also created an out-of-frame ATG triplet (between the nucleotides –2 and +1) and had a phenotype similar to that of pSAM25. To provide a more quantitative data on the mitochondrial complementation activity of these mutants, a growth curve assay was subsequently carried out in YPG broth. Consistent with the observations made from the complementation assay (Fig. 3C), transformants carrying pSAM33 (with ATG9 inactivated) or pSAM20 (with the wild-type gene) grew well in YPG broth, whereas growth of those carrying pIVY100 (with ATG1 inactivated), pSAM25 (with an out-of-frame mutation between the two ATG codons), or pIVY104 (with both ATG triplets inactivated) was severely impaired (Fig. 3D). Analysis of the relative levels of specific mRNAs generated from each of these constructs indicated that similar levels of CaALA1 transcripts were generated from or maintained in the transformants carrying the wild-type or mutant CaALA1 constructs as determined by a semiquantitative RT-PCR experiment (Fig. 3E). This observation suggested that these mutations had little effect on the stability of the specific mRNAs generated from the constructs and, therefore, most likely modulated only the translation initiation activity of the individual initiators. Because the observations made above were a result of AlaRS proteins produced from a high copy number plasmid, we were afraid that they might not accurately reflect protein activity at physiological levels. Therefore, wild-type and mutant CaALA1 genes were cloned into a low copy number shuttle vector (Fig. 4A) and tested for their cytoplasmic and mitochondrial functions. As shown in Fig. 4, B and C, these constructs had complementing activities similar to the corresponding constructs cloned in a high copy number vector. Partition Pattern of ATG1- and ATG9-initiated CaAlaRS Isoforms—To investigate whether the CaALA1 constructs contain similar activities when highly expressed from a constitutive ADH promoter, some of the representative constructs shown in Fig. 3A were subcloned into pADH and tested for their complementation functions. As shown in Fig. 5, A–C, these constructs contained similar complementation functions to those cloned in pRS425, except for the ATG1 mutant, which contained only cytoplasmic function when cloned in pRS425 but contained both cytoplasmic and mitochondrial functions when cloned in pADH (compare pIVY100 and pIVY98). One likely possibility leading to this outcome is that the ATG9-initiated form contains a cryptic mitochondrial targeting signal that normally does not play a role in mitochondrial localization but can be recruited to function when the protein is highly expressed. To directly look at the protein expression levels and elucidate the correlations between complementation functions and partition patterns of the isoforms within the cell, the total, mitochondrial, and cytoplasmic fractions were isolated from each of the transformants harboring various CaALA1 constructs. As shown in Fig. 5D, the proteins expressed from the wild-type CaALA1 construct were partitioned between cytoplasm and mitochondria (lane 1, pIVY97), but mutations that inactivated both of the ATG initiators completely abolished the synthesis of the isoforms (lane 4, pIVY149). Interestingly, when the first ATG initiator was inactivated (lane 2, pIVY98), the protein levels in the total fraction remained almost unchanged, whereas the protein band in the mitochondrial fraction drastically decreased (compare lanes 1 and 2), suggesting that the mitochondrial proteins came largely from initiation at ATG1. In addition, it came as a surprise to us at first to find that the protein levels in the cytoplasmic fraction of this mutant appreciably increased. We surmised that the basis underlying this unexpected observation could probably be attributed to the fact that ATG9 served only as a remedial initiation site in the wild-type construct (lane 1) but became the first available ATG initiator in the ATG1 mutant (lane 2), leading to the higher expression of the “cytoplasmic” form. To assess the initiating activity of the remedial initiation site, i.e. ATG9, more accurately, ATG1 was left unaltered, and two extra nucleotides were inserted into the sequence between the two ATG initiators, causing the first ATG to be out-of-frame with respect to the rest of the open reading frame (lane 5, pIVY152). Under such conditions the protein band in the total fraction only slightly decreased due to loss of the ATG1-initiated protein, whereas the protein level in the cytoplasmic fraction was almost unchanged (compare lanes 1 and 5). But most significant of all, no protein was seen in the mitochondrial fraction of this out-of-frame mutant (lane 5), suggesting that the ATG9-initiated protein form was exclusively confined to the cytoplasm when ATG9 serves only as a remedial initiation site (compare lanes 1 and 5) and could be forced into mitochondria, possibly due to the presence of a cryptic mitochondrial targeting signal, when ATG9 serves as the first available initiator, resulting in higher expression of the short form (compare lanes 2 and 5). By contrast, when the second ATG initiator was inactivated (lane 3; pIVY118), the levels of the proteins in the total or cytoplasmic extracts drastically decreased, suggesting that the cytoplasmic proteins came largely from initiation at ATG9 in the wild-type construct (compare lanes 1 and 3), and the ATG1-initiated protein form can be partitioned in both compartments (lane 3), with the major portion targeted to the mitochondria. It is noteworthy that the protein level in the mitochondrial fraction of this mutant appreciably increased as compared with the wild-type construct (compare lanes 1 and 3). We surmised that perhaps this unexpected increase was due to alterations of the potential MPP cleavage site, which might affect its processing and in turn its distribution. As a control, the mitochondrial and cytoplasmic fractions were also probed with a mixture of antiphosphoglycerate kinase (a cytoplasmic marker protein) and anti-Hsp60 (a mitochondrial marker protein) to check for cross-contamination. As shown in Fig. 5D, no serious cross-contamination was seen in these preparations (lower two panels). To further quantify the initiating activity of ATG1 and ATG9, the relative protein levels in the total fractions of pIVY152 (an out-of-frame mutant) and pIVY118 (an ATG9 mutant) were compared. Fig. 5E showed that the initiating activity of ATG9 is around 4-fold as high as that of ATG1 under the conditions used. As a control, the relative protein levels generated from pIVY97 (wild type) were also shown. This result sugg
e15584 Background: Comprehensive tumor profiling using NGS is fundamentally transforming oncology research. However, converting archival tissue samples into libraries is often challenging due to the low quantity and quality of DNA. Here we present accurate detection of variants in the human exome using the novel chemistry of the xGen Prism DNA library preparation kit, optimized for low-input and degraded samples, with xGen Research Exome v2.0 hybrid-capture enrichment. Methods: The IDT Exome v2 panel was used to carry out targeted sequencing of Prism DNA libraries generated from archival FFPE samples. The unique library preparation is enabled by an engineered mutant ligase and proprietary adapters that prevent chimeras and suppress dimer-formation, thereby maximizing the conversion of input DNA to sequencing libraries. Results: We achieved high yields of library (300-400 ng) from input amounts as low as 25 ng for severely damaged FFPE samples (DIN 1-3), > 90% on-target rates and uniform depth of coverage ( > 96% bases covered at > 20X and > 98% bases covered at > 10X) for FFPE samples across a wide range in quality. We also observed minimal exon drop-outs in difficult-to-target genes for severely damaged FFPE material. To validate the variant calling performance of the Prism-Exome workflow, we used the Horizon OncoSpan FFPE reference control which contains 1-92% AF SNVs and Indels and achieved > 98% sensitivity across ~250 SNVs and Indels. Conclusions: This study demonstrates that the xGen Exome Research v2, when combined with xGen Prism DNA library preparation, provides researchers with a complete human exome FFPE-sequencing solution with robust performance across FFPE samples of varying quality.
tRNAs are the fundamental components of the translation machinery as they deliver amino acids to the ribosomes during protein synthesis. Beyond their essential function in translation, tRNAs also function in regulating gene expression, modulating apoptosis and several other biological processes. There are multiple layers of regulatory mechanisms in each step of tRNA biogenesis. For example, tRNA 3′ trailer processing is altered upon nutrient stress; tRNA modification is reprogrammed under various stresses; nuclear accumulation of tRNAs occurs upon nutrient deprivation; tRNA halves accumulate upon oxidative stress. Here we address how environmental stresses can affect nearly every step of tRNA biology and we describe the possible regulatory mechanisms that influence the function or expression of tRNAs under stress conditions.
266 Background: Clinical validation studies support tumor-informed molecular residual disease (MRD) as a prognostic biomarker for disease recurrence across multiple solid tumor types. However, these tests are not always feasible due to the occasional lack of tumor tissue. Here, we discuss the design of a test for tissue-agnostic MRD detection and its application to a cohort of patients with colorectal cancer (CRC). Methods: A targeted panel composed of differentially methylated regions was developed. A machine-learning model was trained on differential methylation patterns in order to classify plasma samples as MRD-positive or MRD-negative. Performance of the independently trained classifier was assessed in a cohort of 247 patients enrolled in the Bespoke CRC trial (NCT04264702). These patients had MRD results available using a tumor-informed circulating tumor DNA (ctDNA) assay (Signatera), of whom 163 were persistently MRD-negative without clinical progression and 84 had MRD-positive results. Tissue-agnostic MRD results were compared to the tumor-informed results by calculating the percent positive agreement (PPA). Additionally, the differentially methylated allele fraction (DMAF) from the tissue-agnostic test was compared with variant allele frequencies (VAFs) from the tumor-informed test. Results: In the Bespoke CRC clinical cohort (72% non-Hispanic White, 54% male, mean age 61.4±12.3 years), 71 (28%) patients had stage II CRC, and 147 (60%) had stage III CRC. Overall, PPA was 86% (95% CI: 70-100%) and specificity was 97% (95% CI: 93-100%). When categorizing based on tumor-informed MRD VAF levels, PPA was 97% for VAF >0.2%, 100% for VAF 0.1-0.2%, 89% for VAF 0.04-0.1%, and 68% for VAF <0.04%. The DMAFs strongly correlated with the VAFs from the tumor-informed test and the correlation was independent of disease stage, histology, age, and sex. Conclusions: This is the first study of its kind demonstrating high concordance between a tissue-agnostic MRD test and a clinically validated tumor-informed ctDNA assay. These findings demonstrate that in cases where tissue is not available or of inadequate quality, a methylation-based tissue-agnostic assay may serve as a potential alternative for MRD detection.
Abstract Demonstrate performance of a complete automation and reagent workflow for analysis of cfDNA from bodily fluids. The efficient extraction of cfDNA from bodily fluids is a unique challenge due to the very low concentrations of nucleic acid. The extraction process along with library preparation is a laborious workflow, where human variability can lead to increased variability in the downstream analysis. Integrated DNA Technology (IDT) and Beckman Coulter (BC) have teamed up to provide a complete automation and reagent workflow for analysis of low frequency variants in cfDNA. The Apostle MiniMax™ High Efficiency Isolation Kit from BC provides complex, utilized magnetic nanoparticles to effectively capture cfDNA. IDT's library prep kit utilizes novel chemistry to maximize conversion, suppress adapter-dimer formation, reduce chimera rates, and facilitate double strand consensus analysis to call ultra-low frequency variants. Finally, IDT's xGen™ hybrid capture products maintain high library diversity and on-target rates to enable low frequency variant calling regardless of panel size. The Biomek i5 and i7 Hybrid workstations bring out the best performance from these reagents. The Biomek NGS workstations protocol is written with a modular design with safe stop points, making it customizable for each lab. The automated protocol uses Beckman's Demonstrated Method Interface tools which include: Biomek Method Launcher to run the method without going into Biomek software, Method Options Selector to choose the run parameters with a user friendly interface, Guided labware Setup to set the deck with labware based on the run parameters, DeckOptix Final Check software to help reduce deck setup errors. We demonstrate the performance of this complete workflow with a range of plasma inputs (4-8 mL). Using control samples with known variant frequencies, the workflow yields high library complexity, 100% positive predictive value, and reliable detection of <0.5% mutant allele frequency variants. With real cfDNA, the workflow demonstrates both high cfDNA and sequencing library yields along with high library complexity. The combination of these reagents on the Biomek workstations provides a robust and reproducible solution for the analysis of cfDNA. Citation Format: Nicole Roseman, Shilpa Parakh, Hsiao-Yun Huang, Kevin Lai, Timothy Barnes, Lyn Lewis, Ushati Das Chakravarty, Anastasia Potts, Alisa Jackson, Amy Yoder, Jessica Sheu, Tzu-Chun Chen. Improved conversion in extraction, library construction, and capture improve sensitivity for variants in liquid biopsy samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5863.
Many existing pattern recognition techniques require the estimation of the covariance matrix. When the number of available samples is sufficient large relative to the dimension the features, a maximum likelihood estimator or a related unbiased covariance matrix estimator can be applied. In the classification task of the hyperspectral image, however, the number of available observations is very limited or even smaller than the number of bands due to the access of the ground truth samples is costly and valuable. In this case, the performance of the maximum likelihood related estimators will be poor. Thus, the classification accuracy of the corresponding classification methods is unsatisfied. Based on the idea of combining several different structures in an estimator, a new covariance matrix estimator called localized shrinkage covariance estimator (LSCE) is proposed in this study. The performance of LSCE is evaluated via the classification accuracy of the linear discriminant classifier (LDC) using LSCE as the estimator of its covariance matrix. The results of the simulation studies show that LSCE is an ideal covariance estimator and the classical method LDC can be a very competitive classifier comparing to other popular techniques in hyperspectral data classification.
Eukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the cell's DNA replication machinery, most of what we know about cellular DNA replication has come from the study of viral systems. Herein, we focus our study on the assembly of the Kaposi's sarcoma-associated herpesvirus core replication complex and propose a pairwise protein-protein interaction network of six highly conserved viral core replication proteins. A detailed understanding of the interaction and assembly of the viral core replication proteins may provide opportunities to develop new strategies against viral propagation.
Statistical discrimination for nonstationary random processes is important in many applications. Our goal was to develop a discriminant scheme that can extract local features of the time series, is consistent, and is computationally efficient. Here, we propose a discriminant scheme based on the SLEX (smooth localized complex exponential) library. The SLEX library forms a collection of Fourier-type bases that are simultaneously orthogonal and localized in both time and frequency domains. Thus, the SLEX library has the ability to extract local spectral features of the time series. The first step in our procedure, which is the feature extraction step based on work by Saito, is to find a basis from the SLEX library that can best illuminate the difference between two or more classes of time series. In the next step, we construct a discriminant criterion that is related to the Kullback–Leibler divergence between the SLEX spectra of the different classes. The discrimination criterion is based on estimates of the SLEX spectra that are computed using the SLEX basis selected in the feature extraction step. We show that the discrimination method is consistent and demonstrate via finite sample simulation studies that our proposed method performs well. Finally, we apply our method to a seismic waves dataset with the primary purpose of classifying the origin of an unknown seismic recording as either an earthquake or an explosion.
Abstract Independent coffee shops are the alternative workplaces for people working remotely from traditional offices but are not concerned about their indoor air quality (IAQ). This study aimed to rank the environmental factors in affecting the IAQ by Random Forests (RFs) models. The indoor environments and human activities of participated independent coffee shops were observed and recorded for 3 consecutive days including weekdays and weekend during the business hours. The multi-sized particulate matter (PM), particle-bound polycyclic aromatic hydrocarbons (p-PAHs), total volatile organic compounds (TVOCs), CO, CO 2 , temperature and relative humidity were monitored. RFs models ranked the environmental factors. More than 20% of the 15-min average concentrations of PM 10 , PM 2.5 , and CO 2 exceeded the World Health Organization guidelines. Occupant density affected TVOCs, p-PAHs and CO 2 concentrations directly. Tobacco smoking dominated PM 10 , PM 2.5 , TVOCs and p-PAHs concentrations mostly. CO concentration was affected by roasting bean first and tobacco smoking secondly. The non-linear relationships between temperature and these pollutants illustrated the relative low concentrations happened at temperature between 22 and 24 °C. Tobacco smoking, roasting beans and occupant density are the observable activities to alert the IAQ change. Decreasing CO 2 and optimizing the room temperature could also be the surrogate parameters to assure the IAQ.
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