- Email: [email protected]
Coding in noncoding frames
TECHNICAL
TIP
Different methods have been described for the purificaticmof DNAproducts,h~ ahne~ all d ~ ~ ~ ~ d ~ subntances.These comamimmscan interfe~rein the sequeadng nmctitmand the a ~ p h eften ,~,owsdiffereat ~ . The PCR wocedu~ requitesthe use of eazyngs, buffe~sand ~ so these nnm ~ ~ ~ ~ ~ We ~ ~ this microcon~e~ratofsystembecause it is easy and quidc AncXherirnlmrtamadwantageis tl~ omt of the systemdescrilx'd here ¢3 US dotlar~ per mactitm). We ~ a dear favorable quality:time ratiofor the " or sequendng systemin tim aralys/s¢ffunlmownmutations.In dfffictfltatses, such as heterozygousbase detection, we suggest~ by ckming. Desp/mthe exismm:eof a wide range of siral~e altero nadvesZ-4, we be~-ve that this method could be app~ed in hboratories w~h ~tle exlxtleta~ and at ~ ~ ~ ACKNOW1.EDGD~EN'~
D.P. is supported by grants from AIRCand AIDS(ISS-ROMA).The wol'k of J.N. is supported h~rthe Institutde Recerca de H-lospital de Sam Pau. Rmm~c~ 1 Nomdedeu,J.F. et al. (1995) Leukem/a9, 229-230 2 Green, A., 1 ~ , A. and Vaudin, M. (1990) NudeicAcidsRes. 18, 6163-6164 3 Bachman,B., Lueke, W. and Hunsmann,G. (1990) NudeicAcidsRes. 18,1309 4 Khomna,S., Gagel, ILK and Cote, G.J. (1994) Nucleic Acids Res. 22, 3425-3426 Contributed by D. Petmni, C. Pastore, DipartOnento dt Saenze Btoraediche e Ow-ologia llman~ Un#.,e~ita"di Torino, Italia and A. La~, J. $oler, M. Ba~et and J. Nonuled~, Del~rtament d'Hematologia Hospftat de Sam l-ar¢ Baraelona, Sl~m.
(
LETTER
/ ~ Codiningnoncodingframes, - ~ Prokaryotes and viruses frequently code in overlapping frames, and out-of-frame translation also occurs in eukaryotes. How should this influence the way evolutionary pressure acts on DNA? Evolution is generally considered to remodel the in-frame sequences regardless of open reading frames lying in noncoding frames. According to the common view, stop codons in the out-of-frame sequences help to stop mistaken translation in irrelevant reading frames. In reality, evolution might have a much more active control on noncoding frames. As suggested by the following considerations, systematic investigation of noncoding frames would help to identify unrecognized relationships and to elucidate the underlying genetics of DNA. Coding in overlapping frames is a clever way to save space in short genomes and to keep different genes under the same regulatory control. It is frequently present in viruses, bacteria and yeast. In ,,uch organisms '...misreading events can be relied on as a necessary step in natural translation '1. Exchange of genetic
material often occurs between viruses and their eukaryotic hosts. thereby generating overlapping frames in eukaryotic genes. Frameshifts have been found in almost 500/oof colon carcinomas'. In these cases, the translated products act as tumor-associated antigens. However, out-of-frame translation in eukaryotes is not an exclusively pathological phenomenon. Overlapping reading frames have been found in several eukaryotic genes that are subiect to alternative splicing ~5. Stop codons are frequent in overlapping messages. Their role is presumably to interrupt out-offrame translation. Nevertheless. long overlapping reading frames free of stop codons do occur. In a number of cases, the predicted sequences of the hypothetical products of noncoding frames show high similarity to existing expressed proteins. The very high statistical significance of these observations suggests expected, as well as :~_nex~r~'2ctcd, evolutionary relationships('.7. Further, recent evidences suggest that selenoprote~s "rIG MAY 1996 VOL. 12 No. 5 t
Pll ~H~t8-9525~,~20c1~4,
Lo
might be encoded in the +1 reading frame overlapping the human genes encoding CD4, CD8 and HLA-DR (Ref. 8). Overlapping coding frames in eukaryotes might be currently inactive because they represent the !egacT of a primordial phenomenon, extinguished in eukaryotes. As an alternative hypothesis, overlapping frames might be a novel evolutionary strategy established in viruses, currently in the process of being applied to euk,aryotes. This would also explain why they are not expressed. A third hypothesis ks that the observed similarity of hypothetical products of noncoding frames to real sequences, as well as the presence of coding features in noncoding frames, might be random events, as discussed in a recent issue of Trends in Genetics 9. This is an h-itriguing poim because it is not really clear how to measure the randomness of molecular events occurring in out-of-frame products. The evolutionary, pressure is somewhat different in remodelling out-of-frame products, as compared with in-frame products. The statistical methods currently available for the analysis of in-frame
LETTER products might n e e d to be modified w h e n applied to out-of-frame products 10-12. Although recognized in viruses a n d prokaryotes, the coding features of n o n c o d i n g frames have not been generalized in evolutionary terms, nor discussed as a specific evolutionary strategy. U n k n o w n evolutionary links will b e identified by developing appropriate statistics a n d software for systematic investigation of the products of all six reading frames of a n y k n o w n gene. These investigations will clarify the relationships between the DNA
sequence a n d its meaning at the protein level.
ANTONIOF A ~ [email protected] RaggioItalgene, Pomezia. 00040 Roma: and IDI-IRCCS,Lab. Patologia Vascolare via dei Monti di Creta 104, 00167 Roma, Italy.
References 1 Lewin, B. (1994) in Ge,~tes 5. p. 225, Oxford Universiw Press 2 Townsend, A. et aL (1994) Xaturv 371,662 3 lin, B. et al. (199-3) Genomics 25, 93-~) 4 Cascino, I. etal. (1995)J. lmmunol.
154, 2706-2713 5 Languino, L.R. and Ruoslahti, E. (1992) J. Biol. Chem. 267, 7116-7120 6 Facchiano, A. (1995)J. MoL EL~I. 40, 570-57I 7 Facchiano, A. etal. (1993).L MoL Evol. 36, 448-457 8 Taylor, E.W. (1995) Biol. Trace E4em. Res. 49, 85-95 9 Cebrat, S. and Dudek, M.R. (1996) Trends Genet. 12, 12 10 Kadin, S. and Brendel, V. (1992) Science 257, 39-49 11 Kadin, S. and Altschul, S.F. G990) Proc. Nail Acad. Sci. USA 87, 2264-2268 12 Pascarella, S. and Argos, P. (1992) J. Mol. Biol. 224, 461-471
MONITOR
A 'digest' o f s o m e recent p a p e r s o f interest in the p r i m a r y journals.
Evolutionof gene familiesand relationshipwith organismal evolution:rapiddivergenceof tissue-specificgenes in the early evolutionof chordates N. IWABF,K-1.KUMAANt)T. MIYATA MOI. Biol. F.vol. 13, 483-493
It is as yet unclear how far molecular evolution is linked to organismal evolution. If most molecular changes are neutral, one would not expect that the average rates of molecular evolution would be much influenced by the rates of cladogenesis, lwabe et aL have analysed the evolutionary characteristics of 25 different gene families from
The quakinggene productnecessary in embryogenesisand myelination combinesfeaturesof RNAbindingand signal transductionproteins T.A. EBERSOLEETAL ?,'at. Genet. 12, 260-265
This paper illustrates the problem of analysing a gene that is responsible for a pleiotropic mutation. The deletion mutation quaking viable (qk v) arose spontaneously on chromosome 17 and causes a recessive defect affecting myelination in mice. Paradoxically, four lethal ethylnitrosourea (ENU)-induced alleles (qk e) arrest embryonic development before the onset of myelination.
a range of different taxa. These gene families were chosen Ix?cause their members have undergone several rounds of duplication and functional diversification. Using the sequences from the databases, lwabe et al. have produced alignments and molecular phylogenetic reconstructions for each of these families. They show that duplications that have given rise to different functions, such as ion pumps with specificity for different ions, have mainly occurred in the early times of evolution (i.e. beh~re the Cambrian radiation). After ttmt time, duplications have mainly resulted in isofomls with tissue-specific distributions. Such duplications have taken place independently in the major
phyla, such as chordates and arthropods. Within the chordate lineage, the duplications took place mainly soon after the Cambrian radiation, at a time of rapid cladogenesis of the chordates. Most interestingly, not only was the rate of gene duplication enhanced during this time, but also the rate of amino acid substitution. Thus, rapid morphological evolution was accompanied by rapid molecular evolution. Whether this was owing to relaxed constraints - as the authors suggest - or to adaptation effects remains open. This study underlines the importance of using species from many taxa, rather tlxan a few model organisms in comparative sequence studies.
Complementation of the qk e alleles by qk ~' for their lethality, but not for the myelination defect, implicated the role of a single gene with functkmally distinct elements, or two ~ p a m t e but interacting genes. Cloning of the qk v deletion breakpoint identified two classes of multiple transcripts (qk t and q k tt) from a mouse brain cDNA library. Linking of the transcripts IW 3" RACE suggested they were products of the same gene. q k I transcripts feature a number of domains, one of which is homologous to the RNA-binding KH domain of tire mouse (and human) S a m 6 8 gene, and the Caenorbabditis elegans turnout suppressor gene, gld-1. Further regions of qk t homology to
S a m 6 8 suggest QKI could also have signal transduction properties, qkttran scripts showed no alteration in the qk v mutation, but in an ENU-induced allele contained a significant amino acid change in a conserved domain adjacent to the KH motif, q k t! transcripts were present at wild-type levels in qkVhomozygotes but were truncated in size, whilst the qk t transcripts remained unaltered. In the absence of a definitive open reading frame, the function of the q k tt transcripts remains unknown, although it1 $iltt analysis showed q k ! to be expressed in mid-gestation embryos, while Ix~th transtxipt classes were expressed in postnatal brain during periods of myelination.
TIG MAY 1996 VOL. 12 NO. 5
169
TIP
Different methods have been described for the purificaticmof DNAproducts,h~ ahne~ all d ~ ~ ~ ~ d ~ subntances.These comamimmscan interfe~rein the sequeadng nmctitmand the a ~ p h eften ,~,owsdiffereat ~ . The PCR wocedu~ requitesthe use of eazyngs, buffe~sand ~ so these nnm ~ ~ ~ ~ ~ We ~ ~ this microcon~e~ratofsystembecause it is easy and quidc AncXherirnlmrtamadwantageis tl~ omt of the systemdescrilx'd here ¢3 US dotlar~ per mactitm). We ~ a dear favorable quality:time ratiofor the " or sequendng systemin tim aralys/s¢ffunlmownmutations.In dfffictfltatses, such as heterozygousbase detection, we suggest~ by ckming. Desp/mthe exismm:eof a wide range of siral~e altero nadvesZ-4, we be~-ve that this method could be app~ed in hboratories w~h ~tle exlxtleta~ and at ~ ~ ~ ACKNOW1.EDGD~EN'~
D.P. is supported by grants from AIRCand AIDS(ISS-ROMA).The wol'k of J.N. is supported h~rthe Institutde Recerca de H-lospital de Sam Pau. Rmm~c~ 1 Nomdedeu,J.F. et al. (1995) Leukem/a9, 229-230 2 Green, A., 1 ~ , A. and Vaudin, M. (1990) NudeicAcidsRes. 18, 6163-6164 3 Bachman,B., Lueke, W. and Hunsmann,G. (1990) NudeicAcidsRes. 18,1309 4 Khomna,S., Gagel, ILK and Cote, G.J. (1994) Nucleic Acids Res. 22, 3425-3426 Contributed by D. Petmni, C. Pastore, DipartOnento dt Saenze Btoraediche e Ow-ologia llman~ Un#.,e~ita"di Torino, Italia and A. La~, J. $oler, M. Ba~et and J. Nonuled~, Del~rtament d'Hematologia Hospftat de Sam l-ar¢ Baraelona, Sl~m.
(
LETTER
/ ~ Codiningnoncodingframes, - ~ Prokaryotes and viruses frequently code in overlapping frames, and out-of-frame translation also occurs in eukaryotes. How should this influence the way evolutionary pressure acts on DNA? Evolution is generally considered to remodel the in-frame sequences regardless of open reading frames lying in noncoding frames. According to the common view, stop codons in the out-of-frame sequences help to stop mistaken translation in irrelevant reading frames. In reality, evolution might have a much more active control on noncoding frames. As suggested by the following considerations, systematic investigation of noncoding frames would help to identify unrecognized relationships and to elucidate the underlying genetics of DNA. Coding in overlapping frames is a clever way to save space in short genomes and to keep different genes under the same regulatory control. It is frequently present in viruses, bacteria and yeast. In ,,uch organisms '...misreading events can be relied on as a necessary step in natural translation '1. Exchange of genetic
material often occurs between viruses and their eukaryotic hosts. thereby generating overlapping frames in eukaryotic genes. Frameshifts have been found in almost 500/oof colon carcinomas'. In these cases, the translated products act as tumor-associated antigens. However, out-of-frame translation in eukaryotes is not an exclusively pathological phenomenon. Overlapping reading frames have been found in several eukaryotic genes that are subiect to alternative splicing ~5. Stop codons are frequent in overlapping messages. Their role is presumably to interrupt out-offrame translation. Nevertheless. long overlapping reading frames free of stop codons do occur. In a number of cases, the predicted sequences of the hypothetical products of noncoding frames show high similarity to existing expressed proteins. The very high statistical significance of these observations suggests expected, as well as :~_nex~r~'2ctcd, evolutionary relationships('.7. Further, recent evidences suggest that selenoprote~s "rIG MAY 1996 VOL. 12 No. 5 t
Pll ~H~t8-9525~,~20c1~4,
Lo
might be encoded in the +1 reading frame overlapping the human genes encoding CD4, CD8 and HLA-DR (Ref. 8). Overlapping coding frames in eukaryotes might be currently inactive because they represent the !egacT of a primordial phenomenon, extinguished in eukaryotes. As an alternative hypothesis, overlapping frames might be a novel evolutionary strategy established in viruses, currently in the process of being applied to euk,aryotes. This would also explain why they are not expressed. A third hypothesis ks that the observed similarity of hypothetical products of noncoding frames to real sequences, as well as the presence of coding features in noncoding frames, might be random events, as discussed in a recent issue of Trends in Genetics 9. This is an h-itriguing poim because it is not really clear how to measure the randomness of molecular events occurring in out-of-frame products. The evolutionary, pressure is somewhat different in remodelling out-of-frame products, as compared with in-frame products. The statistical methods currently available for the analysis of in-frame
LETTER products might n e e d to be modified w h e n applied to out-of-frame products 10-12. Although recognized in viruses a n d prokaryotes, the coding features of n o n c o d i n g frames have not been generalized in evolutionary terms, nor discussed as a specific evolutionary strategy. U n k n o w n evolutionary links will b e identified by developing appropriate statistics a n d software for systematic investigation of the products of all six reading frames of a n y k n o w n gene. These investigations will clarify the relationships between the DNA
sequence a n d its meaning at the protein level.
ANTONIOF A ~ [email protected] RaggioItalgene, Pomezia. 00040 Roma: and IDI-IRCCS,Lab. Patologia Vascolare via dei Monti di Creta 104, 00167 Roma, Italy.
References 1 Lewin, B. (1994) in Ge,~tes 5. p. 225, Oxford Universiw Press 2 Townsend, A. et aL (1994) Xaturv 371,662 3 lin, B. et al. (199-3) Genomics 25, 93-~) 4 Cascino, I. etal. (1995)J. lmmunol.
154, 2706-2713 5 Languino, L.R. and Ruoslahti, E. (1992) J. Biol. Chem. 267, 7116-7120 6 Facchiano, A. (1995)J. MoL EL~I. 40, 570-57I 7 Facchiano, A. etal. (1993).L MoL Evol. 36, 448-457 8 Taylor, E.W. (1995) Biol. Trace E4em. Res. 49, 85-95 9 Cebrat, S. and Dudek, M.R. (1996) Trends Genet. 12, 12 10 Kadin, S. and Brendel, V. (1992) Science 257, 39-49 11 Kadin, S. and Altschul, S.F. G990) Proc. Nail Acad. Sci. USA 87, 2264-2268 12 Pascarella, S. and Argos, P. (1992) J. Mol. Biol. 224, 461-471
MONITOR
A 'digest' o f s o m e recent p a p e r s o f interest in the p r i m a r y journals.
Evolutionof gene familiesand relationshipwith organismal evolution:rapiddivergenceof tissue-specificgenes in the early evolutionof chordates N. IWABF,K-1.KUMAANt)T. MIYATA MOI. Biol. F.vol. 13, 483-493
It is as yet unclear how far molecular evolution is linked to organismal evolution. If most molecular changes are neutral, one would not expect that the average rates of molecular evolution would be much influenced by the rates of cladogenesis, lwabe et aL have analysed the evolutionary characteristics of 25 different gene families from
The quakinggene productnecessary in embryogenesisand myelination combinesfeaturesof RNAbindingand signal transductionproteins T.A. EBERSOLEETAL ?,'at. Genet. 12, 260-265
This paper illustrates the problem of analysing a gene that is responsible for a pleiotropic mutation. The deletion mutation quaking viable (qk v) arose spontaneously on chromosome 17 and causes a recessive defect affecting myelination in mice. Paradoxically, four lethal ethylnitrosourea (ENU)-induced alleles (qk e) arrest embryonic development before the onset of myelination.
a range of different taxa. These gene families were chosen Ix?cause their members have undergone several rounds of duplication and functional diversification. Using the sequences from the databases, lwabe et al. have produced alignments and molecular phylogenetic reconstructions for each of these families. They show that duplications that have given rise to different functions, such as ion pumps with specificity for different ions, have mainly occurred in the early times of evolution (i.e. beh~re the Cambrian radiation). After ttmt time, duplications have mainly resulted in isofomls with tissue-specific distributions. Such duplications have taken place independently in the major
phyla, such as chordates and arthropods. Within the chordate lineage, the duplications took place mainly soon after the Cambrian radiation, at a time of rapid cladogenesis of the chordates. Most interestingly, not only was the rate of gene duplication enhanced during this time, but also the rate of amino acid substitution. Thus, rapid morphological evolution was accompanied by rapid molecular evolution. Whether this was owing to relaxed constraints - as the authors suggest - or to adaptation effects remains open. This study underlines the importance of using species from many taxa, rather tlxan a few model organisms in comparative sequence studies.
Complementation of the qk e alleles by qk ~' for their lethality, but not for the myelination defect, implicated the role of a single gene with functkmally distinct elements, or two ~ p a m t e but interacting genes. Cloning of the qk v deletion breakpoint identified two classes of multiple transcripts (qk t and q k tt) from a mouse brain cDNA library. Linking of the transcripts IW 3" RACE suggested they were products of the same gene. q k I transcripts feature a number of domains, one of which is homologous to the RNA-binding KH domain of tire mouse (and human) S a m 6 8 gene, and the Caenorbabditis elegans turnout suppressor gene, gld-1. Further regions of qk t homology to
S a m 6 8 suggest QKI could also have signal transduction properties, qkttran scripts showed no alteration in the qk v mutation, but in an ENU-induced allele contained a significant amino acid change in a conserved domain adjacent to the KH motif, q k t! transcripts were present at wild-type levels in qkVhomozygotes but were truncated in size, whilst the qk t transcripts remained unaltered. In the absence of a definitive open reading frame, the function of the q k tt transcripts remains unknown, although it1 $iltt analysis showed q k ! to be expressed in mid-gestation embryos, while Ix~th transtxipt classes were expressed in postnatal brain during periods of myelination.
TIG MAY 1996 VOL. 12 NO. 5
169