The Real Life of Pseudogenes

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48 SCIENTIFIC AMERICAN AUGUST 2006 COPYRIGHT 2006 SCIENTIFIC AMERICAN, INC. Disabled genes, molecular relics scattered across the human genomic landscape, have a story of their own to tell. And it is still unfolding The Real Life of Pseudogenes

By Mark Gerstein and Deyou Zheng

ur genetic closet holds skeletons. The bones of long-dead genes— known as pseudogenes—litter our chromosomes. But like other fossils, they illuminate the evolutionary history of today’s more familiar forms, and emerging evidence indicates that a few of O these DNA dinosaurs may not be quite so dead after all. Signs of activity among pseudogenes are another reminder that although the project to sequence the human genome (the complete set of genetic information in the nuclei of our cells) was officially finished, scientists are still just beginning to unravel its complexities. It is already clear that a whole genome is less like a static library of information than an active computer operating system for a living thing. Pseudogenes may analogously be vestiges of old code associated with de- funct routines, but they also constitute a fascinating record contained within the overall program of how it has grown and diversified over time. As products of the processes by which genomes remodel and update themselves, pseudogenes are providing new insights into those dynamics, as well as hints about their own, possibly ongoing, role in our genome. DAVE CUTLER STUDIO

www.sciam.com SCIENTIFIC AMERICAN 49 COPYRIGHT 2006 SCIENTIFIC AMERICAN, INC. Copied, Not Fake PSEUDOGENE BIRTH AND GENE DEATH “false” genes, which look like real genes but have no apparent function, Two distinct processes can duplicate genes, and together they allow genomes to grow and were fi rst recognized and dubbed pseu- diversify over evolutionary time. If errors in a copy destroy its ability to function as a gene, dogenes during the late 1970s, when however, it becomes a pseudogene instead (right). The mutations that can kill a gene (below) early gene hunters began trying to pin- range from gross deletions (such as the loss of the promoter region that signals the start of point the chromosomal locations associ- a gene sequence) to minute changes in the DNA sequence that skew the meaning of the gene’s ated with production of important mol- protein-encoding segments, called exons. ecules. For example, while seeking the gene responsible for making betaglobin, GENE DEATH a key component of the hemoglobin pro- Genes die and become pseudogenes when can alter their amino acid meaning, and base tein that transports oxygen through the mutations generated during the gene-copying deletions or insertions can affect neighboring bloodstream, scientists identifi ed a DNA process or accumulated over time render them codons by shifting the cellular machinery’s sequence that looked like a globin gene incapable of giving rise to a protein. Cellular reading frame. The alignment shown here of machinery reads the DNA alphabet of nucleotide a partial sequence for a human gene (RPL21) but could not possibly give rise to a pro- bases (abbreviated A, C, G, T) in three-base against one of its pseudogene copies tein. Essential functional parts of the increments called codons, which name an amino (RPL21), along with each codon’s corres- gene’s anatomy were disabled by muta- acid building block in a protein sequence or ponding amino acid (AA), illustrates some of encode “stop” signals indicating the end of a the disabling mutations typically found tions, making it impossible for cellular gene. Even single-base mutations in codons in pseudogenes. machinery to translate the gene into a useful molecule. Synonymous Only the far more recent completion Premature stop codon mutation Gross deletion of sequencing projects covering the full genomes of humans and other organ- AA NRV IE H IK H S K SR DS F L KR V K E N DQ K KK E A KE KG T W VQ L K R Q P A PP RE A H FV R RPL21 A A TG T GC G TA T TG A GC A C A A TA A GC A CT C TA A G A C GC G AG A TA G CT T CC T GA A AC G T G T G A A G G A A A A T G A T C A GA A A A A GA A AG A A G C C A A A G A G A A A G G T A C CT G G G T T C A AC T AA A GC G C C A GC C T G C TC C A C C C A G AG A A G C AC A C T T TG T G A G A isms allowed geneticists to get an aerial view of the genomic landscape and to �RPL21 A A T G T GC A TA T TG A GC A C A T TA A GC A CT C CA A G A C GT G AG A TA A CT C CC T AA A AA A CA T GA A G G A AA A TG A TC A GA A A A A G – – –– – –– – –– – –– – – A A A – G C C A A A G A G T T C A A C T GA A GT G C C A G C C T G C T C T A C C AA G A G A A G T C C A A C T T T G T G A G A AA NHV IE H IK H S K S R D NF L K SS K EN D QK K K Q R V Q L K C Q P A LP RE V F V R appreciate how riddled with such oddi- ties it is. The human genome is made up Nonsynonymous Base deletion and Base insertion and of more than three billion pairs of nucle- mutation frameshift frameshift otides, the building blocks of DNA molecules. Yet less than 2 percent of our genomic DNA directly encodes proteins. With ongoing annotation of the hu- many of them and why, if they are really Perhaps a third is noncoding sequences man genome sequence, our research useless, they have been retained in our within genes, called introns. The re- group, along with others in Europe and genome for so long. maining tracts between genes constitute Japan, have identifi ed more than 19,000 The answer to the fi rst question is al- the great majority of our DNA, and pseudogenes, and more are likely to be ready fairly well understood. A small much of it is effectively genomic dark discovered. Humans have only an esti- fraction of pseudogenes are believed to matter whose function is still largely a mated 21,000 protein-coding genes, so have once been functional genes that mystery. It is in these seemingly barren pseudogenes could one day be found to simply “died” from disabling changes to expanses that most pseudogenes are outnumber their functional counter- their nucleotide sequences. But most randomly scattered like rusted car parts parts. Their sheer prevalence has raised pseudogenes are disabled duplicates of on the landscape—and in surprising many questions, including how they working genes. They may have been numbers. came into existence, why there are so dead on arrival, having suffered lethal damage during the copying process, or Overview/The Pseudogenome they may have accumulated debilitating mutations over time that collectively ■ Pseudogenes are the molecular remains of broken genes, which are unable rendered them incapable of functioning. to function because of lethal injury to their structures. Critical to a working gene is an intact ■ The great majority of pseudogenes are damaged copies of working anatomy that includes uninterrupted genes and serve as genetic fossils that offer insight into gene evolution and nucleotide spans called exons, which genome dynamics. correspond to amino acid sequences in ■ Identifying pseudogenes involves intensive data mining to locate genelike the encoded protein. Introns typically sequences and analysis to establish whether they function. separate the exons, and at the beginning ■ Recent evidence of activity among pseudogenes, and their potential of a gene is a segment known as a pro- resurrection, suggests some are not entirely dead after all. moter that serves as the starting point for cellular machinery to recognize the

FLAWED COPIES Duplication and mutation A “duplicated” pseudogene arises when a cell is replicating Duplicated pseudogene its own DNA and inserts an Promoter Exon Intron extra copy of a gene into the genome in a new location. GENOMIC DNA Gene Processed pseudogene A “processed” pseudogene is formed during gene expression, when a gene is transcribed Transcription into RNA, then that transcript is processed into a shorter messenger RNA (mRNA). Normally, Reverse transcription the mRNA is destined for translation into a RNA transcript and mutation protein—but sometimes it can instead be reverse-transcribed back into DNA form and inserted in the genome. Processing

mRNA Synonymous Premature stop codon mutation Gross deletion

AA NRV IE H IK H S K SR DS F L KR V K E N DQ K KK E A KE KG T W VQ L K R Q P A PP RE A H FV R RPL21 A A TG T GC G TA T TG A GC A C A A TA A GC A CT C TA A G A C GC G AG A TA G CT T CC T GA A AC G T G T G A A G G A A A A T G A T C A GA A A A A GA A AG A A G C C A A A G A G A A A G G T A C CT G G G T T C A AC T AA A GC G C C A GC C T G C TC C A C C C A G AG A A G C AC A C T T TG T G A G A

�RPL21 A A T G T GC A TA T TG A GC A C A T TA A GC A CT C CA A G A C GT G AG A TA A CT C CC T AA A AA A CA T GA A G G A AA A TG A TC A GA A A A A G – – –– – –– – –– – –– – – A A A – G C C A A A G A G T T C A A C T GA A GT G C C A G C C T G C T C T A C C AA G A G A A G T C C A A C T T T G T G A G A AA NHV IE H IK H S K S R D NF L K SS K EN D QK K K Q R V Q L K C Q P A LP RE V F V R

Nonsynonymous Base deletion and Base insertion and mutation frameshift frameshift

gene on a chromosome. When a cell ex- mRNA is copied back into a sequence of nizable because they contain both in- presses a gene, it ﬁ rst recruits essential DNA that is inserted into the genome. trons and exons. Pseudo genes made molecular players to the promoter site, Known as retrotransposition, this phe- from mRNA lack introns and are de- which travel down the gene’s length, nomenon can occur because of the activ- scribed as processed pseudogenes. transcribing it into a preliminary RNA ity of another type of transposable ge- Although the overall distribution of copy. A splicing process next cuts introns netic actor, known as a long interspersed most pseudogenes across human chro- out of the raw transcript and joins ex- nuclear element, or LINE, that behaves mosomes seems completely random, onic sequences to produce an edited mes- like a genomic virus. LINEs carry their certain kinds of genes are more likely to senger RNA (mRNA) version of the own machinery for making DNA copies give rise to pseudogenes. Geneticists or- gene. The mRNA is then read by a ribo- of themselves to insert into the genome, ganize functional genes into families some, a cellular machine that translates and mRNA transcripts that are in the based on their similarity to one another its sequence into the string of amino ac- vicinity when LINEs are active can be in both sequence and purpose. Only ids that forms a protein, the molecule swept up and retrotransposed as well. about a quarter of these family groups that ultimately carries out the gene’s These two processes, duplication are associated with a pseudogene, and function. and retrotransposition, are major forces some families have spun off an inordi- Pseudogenes can be born in two that remodel genomes over the course of nate number of copies. For example, the ways, each of which yields a distinctive evolutionary time, generating new varia- family of 80 human genes that produce facsimile of the original parent gene. Just tion in organisms. They are the means ribosomal proteins has given rise to before dividing, a cell duplicates its en- by which genomes grow and diversify, about 2,000 processed pseudogenes— tire genome, and during that process, an because many replicated genes remain roughly a tenth of the genome’s identi- extra copy of a gene can be inserted into active. But if the gene copy contains dis- ﬁ ed total. In one extreme case, a single the chromosomes in a new location. Al- abling typos or is missing pieces of the ribosomal protein gene known as RPL21 ternatively, a new version of a gene can original, such as the promoter, it will be- has spawned more than 140 pseudogene also be created through reverse tran- come a pseudogene. Those arising from copies.

LUCY READING-IKKANDA scription: during gene expression, the duplication of an entire gene are recog- This disparity probably derives from

8,000 Processed

Human Genome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Human Chromosome

PSEUDOGENE DESCENDANTS (blue) of the ribosomal protein gene preserve mineral fossils. Identiﬁcation of genes and pseudogenes is RPL21 (orange) are scattered across the human chromosomal an ongoing process, but to date more than 19,000 pseudogenes have landscape. Overall distribution of pseudogenes in the human genome been identiﬁed in the human genome—only slightly less than the appears to be completely random, although some local genome current tally of around 21,000 human genes (inset). About 8,000 of regions tend to contain more pseudogenes. Those DNA regions may our pseudogenes are processed; the rest include duplicated be analogous to certain geochemical environments that better pseudogenes and other nonprocessed subcategories.

the activity levels of different genes. can be seen as vast molecular fossil beds from this selection pressure and are free Those responsible for basic cellular offering a silent record of events in our to accumulate all kinds of mutations, in- housekeeping functions, such as the evolutionary past. cluding changes that would be deleteri- genes in the ribosomal protein family, ous to normal genes. Scientists can use are abundantly expressed, providing Family Histories this tendency to derive a kind of molecu- more opportunities to create processed the principles of natural selection lar clock from the nucleotide changes in pseudogenes. appear to extend to individual genes, pseudogenes and use it to study the over-

Because pseudogenes have been ac- strongly constraining mutations in the all dynamics and evolution of the ge- ) creting this way in our genomes for so sequences of functional genes. Beneficial nome. Tracking the evolutionary path of baby long, some are relics of genes eliminated mutations in a gene that improve the genes and pseudogenes helps molecular ( during the course of evolution, and no organism’s fitness therefore tend to be biologists to uncover instances of gene functional version exists today. Others preserved, whereas a sequence change birth and death just as the study of min- are copies of a gene that has so evolved that impairs a gene’s function leads it to eral fossils tells paleontologists about zefa/Corbis over time, the pseudogene’s sequence be discarded. the creation and extinction of species. may reflect an older, earlier version of its Once consigned to the genomic junk Our group has surveyed pseudogenes parent. Consequently, intergenic regions pile, however, pseudogenes are released in the genomes of many forms of life, ); PAUL BARTON mouse

CHROMOSOMES of humans and mice carry MOUSE a very similar array of functional genes Chromosome 14 (orange) but reveal distinct differences in their pseudogenes (blue), which can highlight important turning points in an organism’s evolutionary history. For example, CHRNA2 EPHX2 GULO CLU SCARA3 the counterpart of a mouse gene called Gulo has become a pseudogene (Gulo) in humans and other primates. Gulo makes an enzyme ); DIGITAL VISION/GETTY IMAGES ( that is the last element in a biochemical pathway for synthesizing vitamin C. Most mammals possess the active illustrations gene, but the primate lineage seems CHRNA2 EPHX2 �GULO CLU SCARA3 to have lost it more than 40 million years ago. When the Gulo gene became a pseudogene, primates became dependent on food HUMAN sources of vitamin C to avoid scurvy. Chromosome 8 LUCY READING-IKKANDA (

ranging from bacteria to more complex genes (30 to 40 percent of the OR fami- in the pseudogenes of animals offer hints organisms, such as yeast, worms, flies ly) than rodents or dogs do, suggesting about their diverse life histories that are and mice, and their prevalence across a that some influence has allowed the en- not as easily detected in comparisons of wide range of creatures is striking. The tire ape lineage to get by with a some- working genes, which are strongly con- number of pseudogenes in different ge- what reduced sense of smell. strained by their function. nomes varies greatly, more so than genes, Lancet and his colleagues found in Analysis of the mouse genome, for and it is not readily predictable, because studies of apes, monkeys and other dis- example, has shown that 99 percent of it is neither strictly proportional to the tant primate cousins that the greatest human genes have a corresponding ver- size of a genome nor to the total number loss of olfactory receptor genes—that is, sion in the mouse. Although the human of genes. Comparisons of pseudogenes the greatest increase in OR pseudo- and mouse lineages diverged some 75 in related genomes can nonetheless re- genes—occurred in ape and monkey lin- million years ago, nearly all of the human veal important information about the eages that evolved the ability to see color genome can be lined up against equiva- history of specific genes and the general in three wavelengths of visible light. The lent regions, known as syntenic blocks, workings of molecular evolution. link may suggest that a sensory trade-off in the mouse genome. Yet despite this One of the largest known gene fami- took place over time in the primate lin- similarity in functional genes and over- lies in mammals, for example, consists eage when better eyesight made an acute all genome structure, just a small fraction of more than 1,000 different genes en- sense of smell less critical. of the known human pseudogenes have coding olfactory receptors, the cell-sur- Often, genes involved in an organ- an obvious counterpart in the mouse. face proteins that confer our sense of ism’s response to its environment are What is more, some of the specific smell. Detailed analyses of olfactory re- subject to extensive duplication and di- gene families giving rise to pseudogenes ceptor (OR) genes and pseudogenes by versification over time, leading to large differ significantly between mouse and Doron Lancet and Yoav Gilad of the gene families, such as the olfactory re- human. Using the rate of sequence decay Weizmann Institute of Science in Rehov- ceptor repertoire. Many dead-on-arrival relative to the parent genes to determine ot, Israel, show that humans have lost a pseudogene copies are an immediate by- their age, it is also clear that many pseu- large number of functional olfactory re- product of this process. But the subse- dogenes in the human and mouse ge- ceptor genes during evolution, and we quent death of additional duplicates, nomes have arisen at different times. now have fewer than 500 of them in our which gives rise to new pseudogenes, is These observations indicate that very genome. For comparison, versions of also frequently connected to changes in disparate events have led to independent about 300 human olfactory receptor an organism’s environment or its cir- bursts of retrotransposition that created pseudogenes are still functional genes in cumstances. Consequently, differences pseudogenes in each of the lineages. the genomes of rats and mice. This difference is not surprising giv- MARK GERSTEIN and DEYOU ZHENG are bioinformaticians at Yale University, where Ger- en that most animals depend more for stein is A. L. Williams Professor of Biomedical Informatics and co-director of the Yale their survival on the sense of smell than Program in Computational Biology and Bioinformatics. Zheng, after completing his Ph.D. humans do. In fact, humans have con- at Rutgers University, joined Gerstein’s group in 2003 to begin investigating pseudo- siderably more olfactory receptor pseu- gene activity and evolution. Both authors were initially interested in studying molecular dogenes than chimpanzees do, indicat- structure and simulation, as described in Gerstein’s previous article for Scientific Amer- THE AUTHORS ing that we lost many of those function- ican with Michael Levitt, “Simulating Water and the Molecules of Life” (November 1998). al genes after our split from the ape But Gerstein and Zheng were intrigued by the enormous data analysis challenges posed lineage. Apes, however, have a higher by the sequencing of the human genome and chose to start scanning and sifting the proportion of olfactory receptor pseudo- regions of DNA between genes.

www.sciam.com SCIENTIFIC AMERICAN 53 COPYRIGHT 2006 SCIENTIFIC AMERICAN, INC. Scanning and Sifting clues to whether a mutation is debilitat- Comparison of pseudogenes among studies of pseudogenes in their ing. We can look for premature “stop” genomes has revealed a puzzling phe- own right are really just beginning, be- signs, as well as insertions or deletions nomenon, however: a few pseudogenes cause these fossil genes were long viewed of nucleotides that shift the reading appear to be better preserved than one as little more than a nuisance. Early ef- frame of cellular machinery that decodes would expect if their sequences were forts to catalogue pseudogenes were the gene’s information for making a pro- drifting neutrally. Such pseudogenes largely driven by the need to distinguish tein. These disablements cannot be tol- may therefore be under evolutionary them from true genes when annotating erated by true genes and are thus typical constraint, which implies that they genome sequences. Identifying pseudo- manifestations of pseudogenes. might have some function after all. One genes is not as straightforward as recog- More subtly, the theory of neutral way to try to ascertain whether pseudo- nizing genes, however. Based on charac- evolution introduced by mathematical genes are functioning is to see whether teristic elements, pattern-seeking compu- biologist Motoo Kimura in the 1960s they are transcribed into RNA. Recent ter algorithms can scan DNA sequences holds that nonfunctional DNA sequenc- experiments by Thomas Gingeras of Af- and identify genes with moderate success. es can change freely, without the con- fymetrix and by Michael Snyder of Yale Recognition of pseudogenes, in contrast, straint of natural selection. Thus, indi- University have found that a signiﬁcant relies primarily on their similarity to vidual nucleotide mutations can be di- fraction of the intergenic regions in the genes and their lack of function. Com- vided into two types: those that would human genome are actively transcribed. puters can detect similarity by exhaus- preserve the amino acid sequence of the In their studies, in fact, more than half tively aligning chunks of intergenic DNA protein encoded by a gene, known as the heavily transcribed sequences map against all possible parent genes. Estab- synonymous changes, and nonsynony- to regions outside of known genes. What

lishing a suspected pseudogene’s inabil- mous changes that would alter the mean- is more, a number of those transcrip- ) ity to function is more challenging. ing of the sequence. Because changing a tionally active intergenic areas overlap Just as a living organism can die of protein’s amino acid sequence can abol- with pseudogenes, suggesting that some many different causes, a variety of del- ish its function, a gene under selective pseudogenes may have life left in them. illustration eterious mutations affecting any step in pressure will be more likely to contain Our research group is part of a con- the process of making a protein can dis- synonymous mutations, whereas a non- sortium of laboratories working to unable a copied gene, turning it into a pseu- functional DNA sequence will not be derstand what is going on in the dark dogene. But the sequence itself can offer subject to that constraint. matter of the genome. We are now in the pilot phase of a project to create an “en- cyclopedia of DNA elements” (referred Genes Pseudogenes to as ENCODE) whose ultimate goal is 100 - to identify all of the genome’s parts and their function. Previous studies as well as preliminary ENCODE data indicate 80 - that at least one tenth of the pseudogenes in the human genome are tran- scriptionally active. Knowing that so

ge 60 -

ta many pseudogenes are transcribed does en not tell us their function, but together ); COLORIZATION BY EMILY HARRISON; LUCY READING-IKKANDA ( rc

Pe 40 - with evidence that certain pseudogenes are better preserved than background micrograph

intergenic sequences, it certainly chal- ( 20 - lenges the classical view of pseudogenes as dead. One possibility is that pseudogenes 0 - Human Apes Dog Mouse play some ongoing part in regulating the Olfactory Receptor Gene Repertoire activity of functional genes. Molecular CILIA projecting from human olfactory epithelium (left) are studded with invisible odorant biologists have come to understand in molecule receptors that detect smells. A family of more than 1,000 genes encoding those recent years that many genes in higher olfactory receptors in mammals has been identified, although individual species vary widely organisms do not code for a final protein Virginia Commonwealth University in their total number of olfactory receptor genes and the percentage of that repertoire that product, but instead their RNA tran- has died and become pseudogenes. Large-scale pseudogenization is most often seen among genes that, like the olfactory receptor family, are responsible for responses to the scripts act to control other genes. These environment. An organism’s pseudogenes may therefore reflect species-specific changes regulatory RNA molecules can various- in circumstances during its evolutionary history. ly activate or repress another gene or can

interfere with the translation of that RICHARD COSTANZO

gene’s mRNA transcript into a function- preserving happy accidents or of nature bonuclease enzyme was a pseudogene al protein. And at least two examples of having figured out an efficient way to re- for much of its history but appears to pseudogenes behaving in a similar man- use the broken parts of genes by convert- have been reactivated during recent evo- ner have been documented so far. ing them into regulatory elements. lutionary time. Slight differences in the The first was reported in 1999 by pseudogene complements of individual Michael O’Shea’s research group at the Protogenes people have also been found—for exam- University of Sussex in England. The in- an exciting era of molecular pale- ple, a few olfactory receptor pseudo- vestigators found that in the neurons of ontology is just beginning. We have genes straddle the fence: in most people a common pond snail, both the gene for barely scratched the surface of the pseu- they are pseudogenes, but in some they nitric oxide synthase (NOS) and its re- dogene strata, and once we drill deeper, are intact, working genes. These anoma- lated pseudogene are transcribed into the number of identified pseudogenes lies could arise if random mutation re- RNA but that the RNA transcript of the will most likely grow and we may find versed the disablement that originally NOS pseudogene inhibits protein pro- more surprises. Large-scale pseudogene produced the pseudogene. Might they duction from the transcript of the nor- identification is a very dynamic data- account for individuals’ differing sensi- mal NOS gene. mining process. Current techniques rely tivities to smell? Perhaps, although it is Then, in 2003, Shinji Hirotsune of heavily on sequence comparison to well- too early to guess at the scope or signifi- the Saitama Medical School in Japan characterized genes, and although they cance of this unexpected source of ge- traced deformities in a group of experi- can readily identify recently generated netic variation among humans. mental baby mice to the alteration of a pseudogenes, very ancient and decayed Our studies have suggested, however, pseudogene. The inactivity of an impor- sequences are probably escaping detec- that in yeast, certain cell-surface protein tant regulatory gene called Makorin1 tion. As the sequence and annotation of pseudogenes are reactivated when the had derailed the development of the the human genome itself are refined and organism is challenged by a stressful mice, but Hirotsune had not done any- updated, characterization of pseudo- new environment. Thus, pseudogenes thing to Makorin1. He had accidentally genes will improve as well. may be considered not only as dead disrupted the Makorin1 pseudogene, Recent hints that not all pseudogenes genes (which nonetheless provide fasci- which affected the function of its coun- are entirely dead have been intriguing, nating new insights into our past) but terpart, the Makorin1 gene. and some evidence also exists for the also as potentially unborn genes: a re- Perhaps two dozen examples of spe- possibility of pseudogene resurrection— source tucked away in our genetic closet cific pseudogenes that appear to be active a dead gene turning back into a living to be drawn on in changing circum- in some way—often only in certain cells one that makes a functional protein stances, one whose possible roles in our of an organism—have been identified, al- product. Careful sequence comparisons present and future genomes are just be- though the findings are still preliminary. have shown that one cow gene for a ri- ginning to unfold. Because many pseudogenes have sequences highly similar to those of their MORE TO EXPLORE parent genes, it is very tempting to specu- Human Specific Loss of Olfactory Receptor Genes. Yoav Gilad et al. in Proceedings of the National late that the NOS and Makorin1 pseudo- Academy of Sciences USA, Vol. 100, No. 6, pages 3324–3327; March 18, 2003. genes are not just isolated cases. Yet it is Pseudogenes: Are They “Junk” or Functional DNA? Evgeniy S. Balakirev and Francisco J. Ayala hard to imagine that these two pseudo- in Annual Review of Genetics, Vol. 37, pages 123–151; December 2003. genes had the specific roles they now per- Large-Scale Analysis of Pseudogenes in the Human Genome. Zhaolei Zhang and Mark Gerstein form when they first arose. Instead their in Current Opinion in Genetics & Development, Vol. 14, No. 4, pages 328–335; August 2004. activity may be the result of selection www.pseudogene.org/