The evolution of a complex eucaryotic gene

The Evolution

of a Complex

Bert W. O’Malley, Two

thirds

of the

surrounding however,

natural

junctions.

the sequences

surrounding

the other

a primordial

of the ovomucoid

ovomucoid

were

Finally,

the positions

during

are unambiguous.

present

for functional the process

in the

gene

of the introns domains

by two

primordial

The three

has

P. Stein, and Anthony been

junction

contain

sequences.

separate

This analysis

intragenic gene

the ovomucoid and provide

birds

gene support on the

all exons four

and

of the

and thus

to the GT-AG

sequence

to examine

suggests

that

the theory manner

that

introns

genes

redundant;

sites

We

at either

compare

the

of the evolution

300

separate

sequences

are

gene

analyses

about

eucaryotic

intron

ovomucoid

sequence

diverged,

by which

rule.

theories

the present

Furthermore,

and mammals

the

introns

the splicing

conforms

duplications.

before

insight

including

no redundancies

protein

Gene

R. Means

surrounding

in all cases

the ovomucoid

suggest

million

gene

years

segments

were

of

evolved that ago. that

constructed

of evolution.

T

HE ORGANIZATION of eucaryotic genes has been an area of acute interest to us for several years. One particularly attractive model system for studying the structural organization of functionally related genes in a single tissue has been the hen oviduct.’ The chicken ovalbumin gene has been cloned and sequenced.*” More recently, the chicken ovomucoid gene was cloned and partially sequenced.’ ” We discovered that both of these oviduct genes exhibited a surprisingly complex structure; each gene contained seven nontranslated regions of DNA sequence (intervening sequences or introns) interspersed among eight genomic DNA regions (exons) that code for the mature polypeptide chains. A number of laboratories in addition to our own have identified the existence of these intervening sequences in diverse eucaryotic genes. ‘* *’ Even though the existence of these intervening sequences seems to be the rule rather than the exception in eucaryotic genes, their function and origin remain an intriguing mystery. The ovomucoid gene afforded us a unique opportunity to examine the raison d’etre of intervening sequences. We have sequenced most of the chicken ovomucoid gene, including all of the intron/exon junctions.” During the course of our investigation, the sequence of the secreted chicken ovomucoid protein was published.29 We are thus able to analyze the structure of the ovomucoid gene in light of the protein sequence. The results of this analysis, which we present here, have allowed us to propose a theory of the function of intervening sequences in the organization of the ovomucoid gene. This in turn has led us to

From the Department of Cell Biology. Baylor College of Me& tine, Houston, Texas, Division of Endocrinology, University of

Texas Health Science Center, Houston, Texas. Address reprint requests to Bert W. O’Malley. Department sf Cell Biology, Baylor College of Medicine. Houston. Texas 77030. @ (982 by Grune & Stratton. Inc. 0026-0495/82/3107~002$01.00/0

646

sequenced, sequences

introns

The splicing

ovomucoid

within

of proteins

gene

gene with

as well as the origin of intervening

introns code

introns

organization

ovomucoids from

ovomucoid

intron/exon

end of these three structural

chicken

all fourteen

Joseph

Eucaryotic

propose a mechanism eucaryotic genes.

for the evolution

of complex

‘The nucleotide sequence of ovomucoid mRNA was determined from two recombinant DNA plasmids. pOM 100 and pOMS02. (These recombinant plasmids were derived from the parent bacterial plasmid. pBR322; the OM refers to the fact that ovomucoid DNA sequences were inserted, and the three digit numbers simply refer to the chronological isolation sequence.) Preparation of the recombinant plasmid pOMlO0, which contains cDNA synthesized from ;I partially purified mRNA,,,,,. has been described.. This plasmid contained a 650 base pair (bp) DNA,,,,, insert. but lacked - I50 bp corresponding to the 5’ end of mRNA,,,,,. To obtain cDNA sequences containing the 5’ end of the mRNA sequence. a 204 bp fragment removed from the middle of the ovomucoid cDNA sequence in pOMlO0 by cutting with the restriction endonuclease Pst I. was purified and hybridized with ;t partially purified preparation of mRNA,,,,,. DNA complementary to the 5’ end of mRNA,,,>, was synthesized by reverse transcriptase in the absence of an oligo deoxythymidilate primer. The use of a sequence specific primer prevented the polymerization of cDNA from mRNAs other than mRNA,,,. Double-stranded cDNA,,, was prepared and cloned in the Pst I site of pBR322.” The largest resulting recombinant plasmid was designated pOM502. This plasmid contained :I DNA,,,, insert of 538 nucleotides, excluding the CC‘ tails at either end of the cDNA,, sequence. These two plasmids were sequenced, using the method of Maxam and Gilbert.‘” Together, they contained all 821 nucleotides of the cDNA,,,. The ovomucoid structural gene sequence determined from these plasmids is schematically presented in Fig. 1. The 82 I nucleotides that represent the mature ovomucoid mRNA can be divided into four regions. There is a 53

MetaboCsm.Vol.

31,

No

I iJulyl,

1982

647

EVOLUTION OF A COMPLEX EUCARYOTIC GENE

OVOMUCOIDSTRUCTURALGENE

SEQUENCE

82 1 nucleotides 558

I

“Ph

I

&II

SIGNAL

138

1 HPh I

Hph I

5’ LEADER

I

I UQA

.skI

SEQUENCE

PEPTIDE

OVOMUCOID 3’ NONCODING

SEQUENCE

PROTEIN

SEQUENCE

SEQUENCE

Fig. 1. The Ovomucoid Structural Gene Sequence. The lengths of various regions of the mRNA. in nucleotides, are given at the top of the figure. Region 1. the 5’ leader sequence, refers to the portion of the mRNA at the 5’ end that occurs before the AUG initiation signal and hence is not translated into a protein sequence. Region 2. the signal peptide sequence, is that portion of the mRNA which is translated into the signal peptide. the N-terminal portion of the protein which is clipped off during the secretion of the protein. Region 3, the ovomucoid protein sequence, is that portion of the mRNA that codes for the secreted protein sequence. Region 4, the 3’ noncoding sequence, is that portion of the mRNA that follows the UGA termination signal and hence is not translated into a protein sequence.

nucleotide 5’ leader sequence prior to the AUG initiator codon. This is followed by a 72 nucleotide signal peptide sequence, which is translated but removed from the secreted polypeptide during the secretion process.3’ The 558 nucleotides that code for the secreted ovomucoid are next, followed by the 138 nucleotides of 3’ noncoding sequence which are located after the UGA translation termination signal. A poly (A) tail of unspecified length follows this sequence. The cloned ovomucoid structural gene sequences were nick-translated with labeled nucleotides and used to isolate the ovomucoid gene from a library of chicken oviduct DNA fragments. The structural organization of the ovomucoid native gene was then determined by restriction endonuclease, electronmicroscopic mapping, and limited DNA sequence analysis. This physical map of the entire ovomucoid gene is shown in Fig. 2. The gene is 5.6 kilobases (kb) in length, with seven intervening sequences of various lengths interspersed among the eight coding (exon) segments. Remarkably, only 15% of the ovomucoid gene is represented in the mature mRNA.

These junctions demonstrate two different types of splicing sites: those with a redundant sequence at the adjacent junctions and those with no redundancy. There is an extensive terminally redundant sequence surrounding introns A and B and a very limited redundancy surrounding introns F and G. The splice point could occur at any position within the boxes and still yield the correct mature mRNA sequence. Terminally redundant sequences such as these have been found at all splicing sites examined prior to ovomucoid, although the redundant length varied from one to five nucleotides.32 However, the junctions at either end of three ovomucoid introns, C, D, and E, have no redundant sequences. Since the mRNA sequence is known” Structure of the Ovomucoid

71

Gene

AUG

v UGA

A

8

CDE

F

G1

Sequence Analysis of the Ovomucoid Gene

The DNA sequencing method of Maxam and Gilbert” was used to sequence more than 70% of the chicken ovomucoid gene, including all of the exons, the sequences surrounding all fourteen intron/exon junctions, and several complete introns. Because the ovomucoid mRNA was sequenced, we were able to determine exactly where in the coding sequence the introns are inserted. The DNA sequences surrounding these fourteen intron/exon junctions are listed in Fig. 3.

tp

5.6Kbp4

0

Structural

m

Intervening

-

Flanking

Gene Sequence Gene Sequence DNA Sequence

Fig. 2. Structural Organization of the Chicken Ovomucoid Gene. The ovomucoid exon sequences are shown as open bars, the intron sequences es filled bars, and the flanking genomic DNA as e solid line. The seven introns are labeled A through G. The overall length of the ovomucoid gene is 5.6 kb.

O’MALLEY.

sequence

Exon ---

lntron

I

Length 111 -900

A II

20 6

Ill/C

725

TGCCTACAGCATGTGTGTACTGCA

C/IV

TTCCCTCTTCAGlAGAATTTGGAAC

iv/D

GAAACTGTTCCTGTAAGTGAAACC

DIV

CTCCTTCCACAGATGAACTGCAGT

VIE

TGCCCACAAAGTGTTATTGTACCG

E/VI

TTTTCCTTTCAOAGAGCAGGGGGC

VI/F

GCTGCTGTGAG

F/VII

GTGCTTTTGCA

VII/G

CAATGCAGTCG

G/VIII

CCTCGCTTTCAG

TACGTACAGCC AAAGCAACGG

t

STEIN, AND

MEANS

were completing the sequence analysis of the ovomucoid gene, this group published the complete amino acid sequence of the secreted chicken ovornucoid.‘” Furthermore, these investigators noted ;I general sequence homology among avomucoids and several mammalian submandibular and seminal plasma trypsin inhibitors. Specifically. the sequence of chicken ovomucoid shown in Fig. 4, could be divided into three structural domains, I, II, and 111. Each domain ih apparently capable of binding one molecule of trypsin or other serine protease, so there is a good correlation of functional and structural domains. Whereas all three domains contain three disulfide bonds in almost identical positions in each domain, the sequence homology between domains I and II is far stronger than that between domains I and Ill or II and III. Kato ct al.” used these observations to propose that distinct intragenic doubling events of a primordial gene

Fig. 3. DNA Sequences at all Fourteen Intron/Exon Junctions of the Ovomucoid Gene. The DNA sequences at each of the intronlexon junctions ere listed at the left, with exon sequences underlined. The sequences are aligned so that the putative splice points ere above the errow. Terminally redundant sequences at either end of a particular intron are shown in boxes. The actual length of each intron and exon, in nucleotide pairs, is shown at the right.

(underlined in Fig. I), there is only one dinucleotide bond at each junction that could be cleaved and joined with the adjacent exon sequence to yield the correct mature mRNA sequence. In each case, splicing at the indicated position yields an intron whose sequence begins with a GT and ends with an AG dinucleotide. The sizes of the exon sequences are listed in the columns at the right of Fig. 3. These coding segments vary widely in length, the largest, exon VIII, being nine times longer than the shortest, exon II. Exon II is remarkable in that it is only 20 nucleotides in length. This is among the shortest structural gene segments (exons) described to date; only the D gene segment of the immunoglobulin heavy chain gene, which might consist of as few as 13 nucleotides, is as small as exon 11.” The size of such short exons would seem to put a severe limitation on any model of RNA splicing that involves a specific protein and/or RNA interaction with the coding (exon) sequence. The Sequence

of the Secreted

C-terminus

Hen Ovomucoid

Avian ovomucoids are a family of glycoproteins that account for about 10% of the protein content of all bird egg whites.32’33 Chicken ovomucoid has been shown to be responsible for most of the trypsin inhibitory activity of chicken egg white.34 Laskowski and coworkers have for several years been investigating the comparative biochemistry of these avian ovomucoids. While we

Fig. 4. Amino Acid Sequence of the Secreted Chicken Ovomucoid. The single-letter amino acid code by Dayhoff is used. The drawing is meant to depict three similar structural regions held together by disulfide bonds (solid lines) separated by short connecting peptides.

649

EVOLUTION OF A COMPLEX EUCARYOTIC GENE

resulted in the present ovomucoid gene. This hypothesis can now be examined more critically by comparing the ovomucoid genomic DNA sequence with the amino acid sequence of the secreted polypeptide. Ovomucoid Intervening Sequences Functional Domains

DOMAIN _

I

DOMAIN

II

Specify

The illustration of the amino acid sequence in Fig. 4 is depicted so that the functional, trypsin-binding domains of Kato et a1.29are clearly delineated. They are depicted (in one dimension) as globular regions held together by disulfide bonds and separated by short connecting peptides. An analysis of the positions of the intervening sequences within the ovomucoid gene determined that several introns interrupted the portions of the gene sequence that code for the connecting peptides. We used the locations of the introns to redefine the three functional domains of chicken ovomucoid. These domains are depicted separately in Fig. 5. They are aligned to show the structural similarities as well as the similar positions of the introns within the domains. Each domain is separated from the next by one intervening sequence. All domains also contain one internal intervening sequence at identical positions within the three domains. As the lower part of Fig. 5 shows, 46% of the amino acids of domains I and II are identical. Sixty-six percent of the mRNA sequences coding for domains I and II are identical. The homology of domains I and II with domain III is less extensive, but still significant. This suggests that the domains are related, a point which will be discussed in more detail below. Although introns B, D, and F apparently separate the genomic domains coding for the functional peptide domains shown in Fig. 5, what about the other three introns, C, E, and G? These three introns occur at identical positions within the three domains, I, II and III, shown in Fig. 5. These “internal” introns divide the subdomains that code for the trypsin-binding sites, the N-terminal halves of the peptide domains in Fig. 5, from the C-terminal subdomains. It is quite possible that a primordial ovomucoid domain consisted only of the 5’ subdomain, and that the internal introns were created in the course of the addition of the 3’ subdomain. By adding another disulfide bond to the primordial ovomucoid polypeptide (a single domain), this may have resulted in increased stability and increased resistance to digestion by the protease bound by the ovomucoid protein. If these subdomains were separated and the disulfide bridge broken, the functional integrity of the inhibitor would probably be destroyed. Thus, a strong argument can be made that the intervening sequences of the ovomucoid gene divide the gene into structural domains. These genomic domains

InWon E

DOMAIN

Ill

t lntron F

) lntron G % HOMOLOGY

DOMAINS l/II

A.A. 46

N.A. 66

ll/lll

30

42

l/Ill

33

50

Fig. 5. Alignment of the Three Domains of the Secreted Ovomucoid Showing the Similar Positions of Introns. The top portion of the figure shows the secondary structure of the three domains of the secreted ovomucoid polypeptide chain, aligned to emphasize the structural similarities. Disulfide bonds are indicated by the solid lines. The positions of the six introns which interrupt the DNA sequence coding for the three domains are shown with errors. The homology between the amino acid sequence of the domains and the mRNA sequence coding for these domains is shown at the bottom of the figure. The amino acid homologies were calculated by comparing the amino acids at each position of the three domains, beginning with the first amino acid following introns B, D. and F. as shown. The nucleic acid homologies were calculated in the same manner, by comparing each nucleotide that codes for the amino acids shown in the figure. (Therefore, there are three times as many comparisons as for the amino acid homology.) A nine amino acid deletion after the ninth position of domain Ill is assumed to maximize homology with domains I and II.

code for structural domains of the ovomucoid polypeptide, which appear to correspond as well to functional polypeptide domains. We conclude, therefore, that the ovomucoid intervening sequences specify functional domains of the protein, and suggest that this is likely a general concept of eucaryotic gene evolution. Evolution

of the Ovomucoid

Gene

The hypothesis that the present ovomucoid is the result of two intragenic doubling events is quite tenable

650

in light of our investigations of the structural organization of the ovomucoid gene. All three functional polypeptide domains can be aligned so that the secondary structures are remarkably similar. All three domains contain three disulfide bonds in virtually identical positions. Three introns, B, D, and F. delineate the three functional domains of the secreted ovomucoid polypeptide as well as a separate domain that contains the signal peptide. All three domains contain an internal intron at identical positions within the genomic coding sequence. A more detailed analysis of the DNA sequences surrounding the six splice points indicated in Fig. 5 is even more revealing. The splice points (B. D. and F) that separate each domain occur between two codons of the genomic DNA sequence. The internal splice points (C, E, and G) within each domain, however, occur between the second and third nucleotides ofa codon of the genomic DNA sequence. Thus, not only are the positions of the introns within the amino acid sequence the same for all three domains, but the pattern of intron interruption of the codon sequence is also identical. These observations are only plausible if the present ovomucoid gene is the result of two intragenic duplications of a primordial ovomucoid gene which itself contained introns. The primordial ovomucoid gene probably consisted only of the 5’ subdomain of domain I I I lntron G would have been created in the course of addition of the 3’ subdomain, which would have added stability to the primordial protease inhibitor, as discussed above. This recombinational event would have resulted in the creation of the original ovomucoid domain, represented as domain III in Fig. 5. Two genomic duplications, with the retention of noncoding sequence within and at the 5’ end of the primordial gene, would subsequently have led to the present ovomucoid gene. After the initial duplication event, in which domain III gave rise to domain II, there must have been a deletion of twentyseven nucleotides (coding for nine amino acids, see Fig. 5) in the 5’ region of domain 111. The duplication of domain II to form domain I must have occurred much more recently on the evolutionary scale, as judged by the significant homology between the nucleotides at corresponding positions of the genomic sequences coding for domains I and II. An approximate time when this second duplication must have occurred can be estimated by comparing the avian ovomucoids with mammalian protease inhibitors. For example, the dog submandibular protease inhibitor has been sequenced and was shown to consist of two polypeptide domains; the dog domain II (the carboxyterminal domain) shows close homology to the ovomucoid (carboxy terminal) domain III.29 Since all avian species examined to date contain ovomucoids

O’MALLEY.

STEIN. AND MEANS

with three domains, the duplication to form the newest domain ( I ) must have occurred after the divergence of birds from mammals (about 300 million years ago). The evolutionary ancestor to birds and mammals must. therefore, have contained an ancestral ovomucoid with two domains, and we can conclude that the intervening sequences must have existed in the ovomucoid gene well before 300 million and probably over 500 million years ago. Up to this point. our discussion has not included the signal peptidc because it is not part of the secreted ovomucoid, and is, of course, significantly different than the three polypeptide domains. lntron B separates the genomic DNA coding for the bulk of the secreted ovomucoid from the gcnomic domain that codes for the signal peptide and the first two amino acids of the secreted ovomucoid polypeptide. However. intron A separates the DNA coding for the tirst I9 amino acids of the signal peptide from the DNA coding for the remainder of the signal peptide and the first two amino acids of the secreted protein. If each cxon corresponds to a functional or structural domain, then the first 19 amino acids of the ovomucoid signal peptide should comprise a domain that contains the information necessary for translocation, while the remainder of the signal peptide might comprise a domain that contains the peptidasc specificity. In support of this prediction. the initial 14 amino acids which constitute the signal peptide sequence are encoded in exon 1 of the conalbumin gene” as are the first I5 amino acids of the mouse immunoglobin light chain leader sequence.“.” However, this does not appear to be the case for the rat preproinsulin genes. “2 In our evolutionary scheme. the genomic ovomucoid domain containing these first two cxons must have been added to the 5’ end of the primordial ovomucoid gene at the time of the development of the capacity for secretion of the ovomucoid polypeptide. We can now formulate a theory for the evolution ot the ovomucoid gene, and its resultant protein structure, as shown in Fig. 6. The top line shows a single exon sequence. which codes for an ancestral protease inhibitor. The inhibitor was probably only moderately successful as its chosen task, the inhibition of proteases, due to digestion of the inhibitor by the protease. However, with the addition of another appropriate exon to the 3’ end of the original exon (step I). the primordial ovomucoid gene was created, which now coded for a stable inhibitor due to the addition of the strategically located third disultide bond, as discussed above. This polypeptide corresponds to the present day ovomucoid domain 111. Step 2 represents the initial duplication of the primordial ovomucoid gene, perhaps by unequal crossing-over to form an ovomucoid gene

651

EVOLUTION OF A COMPLEX EUCARYOTIC GENE

Ll

Fig. 6. The Proposed Evolution of the Chicken Ovomucoid Gene. The column on the left represents the ovomucoid gene structure at various times during its evolution, and the protein structure for which it codes is shown in the column to the right. The 5’ and 3’ represent the transcription initiation and termination sites, respectively. The solid boxes representexon sequences.Between exons, the thin lines represent intervening sequences; outside of the exons, the thin lines represent untranslated or flanking sequences.

coding for a stable inhibitor with two active sites, domains II and III. It was at this point in evolution, about 300 million years ago, when the avian and mammalian lineages diverged. Mammals have retained this two-domain protease inhibitor, while the avian gene evolved further. Step 3 represents the duplication of domain II, again most likely an unequal crossover event, to create a new domain, the present polypeptide domain I. The result was an ovomucoid gene that codes for the three-domain inhibitor that birds have today. Because all avian ovomucoids examined to date have this three-domain structure,29 the partial genomic duplication of step 3 must have occurred before the avian speciation, or 80-300 million years ago. Step 4 represents the addition of the exons coding for the signal peptide, which created an ovomucoid that could be secreted by the cells responsible for its synthesis (e.g., tubular gland cells of the oviduct). However, as noted in step 1, this addition could have occurred at any point in the evolution of the gene, and indeed must have occurred before the secretion of the protease inhibitor became a necessity. INTRONS AND EUCARYOTIC GENES

In conclusion, our results on the structural organization of the ovomucoid gene have led us to formulate a unified theory of the evolution of the ovomucoid gene. The structure of this gene is consistent with the model

stableblata. 3 Achre

sites

secreted ovomucoid

proposed by Gilbert and ourse1ves38+39*40 that exons may represent protein domains that were “marshalled” together during evolution. Furthermore, we have extended these ideas to make certain predictions concerning the evolution of complex eucaryotic genes in general. Intervening sequences apparently specify structural/functional domains of proteins. This is certainly the case for the ovomucoid, immunog10bin4’ and insulin genes,24 and apparently also for the globin gene.42 The origin and function of intervening sequences cannot be proven, but we can make some educated guesses now as to how and why they arose. We have deduced that the ovomucoid introns are very old; in fact, they probably date from the time when the genes were first formed. As shown in Fig. 6, introns were formed when two exon segments, which already had a built-in function, were brought together to form a primordial gene. The recombinational advantage that these intron sequences afforded is shown in more detail in Fig. 7. Column I shows the problem inherent in the assembly of complex genes from diverse exon segments: the recombination must occur at precise sites at either end of the exons. Otherwise, the reading frame of the resulting mRNA would be altered and the functional integrity of the resultant polypeptide possibly destroyed. Since the recombination process itself is random, a requirement for absolute precision in break-

652

O’MALLEY.

STEIN,

AND

MEANS

Hypothesis for the Origin of lntwwning Sequmes

An EvoUii

Fig.

7.

pothesis vening

Origin

represent

of DNA

lines

introns

or

from

elements

without while

the

DNA

exon

benefit

column

of

segene

diverse

of

II repre-

alternative

generation

thin

either

I shows

assembly

counted

the

flanking

assembly

gene

open regions

while

Column

sents

The

represent

quences.

introns,

Hy-

of Inter-

coding

(exons),

solid

age (or excision) of the segments of DNA to be of low assembled makes the event an occurrence process would be probability. Thus, the evolutionary lengthened greatly. Column II depicts the same event, as we now propose it actually occurred, leading to the development of modern complex eucaryotic genes. Recombination, to bring together the diverse exon segments, could have occurred at any one of numerous sites in the flanking DNA sequence on either side of the exon sequences. This model would greatly facilitate genetic evolution since the probability of occurrence would be orders of magnitude more. In this manner, eucaryotes would be afforded the capacity for greater

the

Sequences.

boxes

(Protein Synthesis)

An Evolutionary for

method in

of

which

the

introns

is

ac-

for.

genetic development per unit time. After rccombination. an intervening sequence would have been created between the two exons. Provided that an early mechanism existed (or developed) for splicing these introns out of the primary RNA transcripts. this process would allow the rapid development of a diverse functional gene. Recent evidence from a number of laboratories has shown that such a splicing enzyme does exist and that introns do not provide a barrier to functional gent expression.” 4XThus, our experimental data and the line of reasoning described above. lead us to believe that intervening sequences are intimately related to the origin and evolution of complex eucaryotic gents.

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EVOLUTION OF A COMPLEX EUCARYOTIC GENE

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