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Proc. Natil. Acad. Sci. USAVol. 81, pp. 3781-3785, June
1984Evolution
Segmental homology and internal repetitiousness identified
inputative nucleic acid polymerase and human hepatitis B
surfaceantigen of human hepatitis B virus
(oligomeric repeats/alternative open reading frame/oligopeptidic
repeats/segmental homology)
SUSUMU OHNOBeckman Research Institute of the City of Hope,
Duarte, CA 91010
Contributed by Susumu Ohno, March 5, 1984
ABSTRACT In a previous paper, it was argued that onlythose
coding sequences descended from oligomeric repeats (thenumber of
bases in the oligomeric unit not being a multiple of3) can retain
sufficiently long alternative open reading frames,and that such
alternative open reading frames serve as the res-ervoir for the
sudden generation of new polypeptide chainswith novel functions. It
was suggested that plasmid-encoded 6-amino hexanoic acid linear
oligomer hydrolase that suddenlyendowed Flavobacterium sp. K172
with the capacity to live offnylon by-products arose by the above
mechanism. A corollaryto the above argument is the expectation that
those viral basesequences that are known to use two of the three
alternativereading frames to encode two different polypeptide
chainsshould invariably contain recognizable remains of the
oligo-meric tandem repeats, and as a consequence, various
oligopep-tidic repeats should also be present in the amino acid
sequenceof each. Furthermore, two polypeptide chains encoded by
thesame base sequence translated in different reading framesshould
show segmental homology of the type depicted previ-ously. In the
present paper, the base sequence of human hepa-titis B virus ayw
subtype that encodes an 832 amino acid resi-due long putative
nucleic acid polymerase in one readingframe and a 226 residue long
human hepatitis B surface anti-gen in the other reading frame was
examined. All three predic-tions noted above were satisfied.
The mechanism of gene duplication as the means to generatea new
gene encoding a functionally novel protein from a re-dundant copy
of the preexisting gene is a rather slow (each1% amino acid
sequence diversification from the ancestralprotein requires roughly
one million years) and ineffectual(most of the redundant copies
become degenerate pseudo-genes) process (1). It thus appears
improbable that thismeans alone sufficed early in the evolution of
living crea-tures, for the requirement of that critical stage was
the al-most simultaneous creation of a sufficiently diverse
varietyof proteins.
Bearing the above in mind, it was proposed that the firstset of
coding sequences that emerged several billion yearsago almost had
to be repeats of various base oligomers. Pro-vided that the number
of bases in the oligomeric unit is not amultiple of three, a high
percentage of the randomly generat-ed oligomeric repeats (e.g.,
59.14% of the monodecamericrepeats) has not one but all three
reading frames open forindefinite lengths. Initially, three
polypeptide chains en-coded by such an oligomeric repeat had to be
identical, butthe accumulation of randomly sustained base
substitutionswould soon have diversified their amino acid
sequences, en-abling certain descendants of each oligomeric repeat
to spec-ify at the most three and more often two functionally
diverse
polypeptide chains simultaneously (2, 3). Certain of the mod-ern
genes retaining enough of the original internal repeti-tiousness
should maintain a long alternative open readingframe, and such
unused open reading frames may serve asthe reservoir from which new
proteins with novel functionscan be generated in one fell swoop. As
an example, it wassuggested that the plasmid-encoded enzyme 6-amino
hexa-noic acid linear oligomer hyt,;-olase, which conferred the
ca-pacity to metabolize nylon !-y-products to Flav'obacteriurmsp.
K172 within 30 years of its encounter with man-madenylon (4-6), was
derived from an unused open reading frameof the coding sequence
that originally specified a 472 aminoacid residue long
arginine-rich protein (3).
In the exceptionally small genome of certain viruses,
twoalternate open reading frames of the same base sequence areoften
used to yield two polypeptide chains of dissimilar func-tions. The
customary explanation that the very smallness oftheir genome
dictated such an efficient utilization of thesame base sequence is
in reality a mere a posteriori state-ment constituting no
explanation at all, for natural selectionis bound to ignore an
unused open reading frame of the exist-ing coding sequence until it
too begins to specify a polypep-tide chain of relevant function. It
follows then that, unless agiven base sequence was a priori endowed
with more thanone translatable open reading frame, the protective
surveil-lance by natural selection could not have covered both of
itsreading frames.
In this paper, the portion of the human hepatitis B viralgenome
that encodes an 832 amino acid residue long putativeDNA polymerase
(or reverse transcriptase?) in one readingframe and a 226 amino
acid residue long human hepatitis Bsurface antigen (HBS antigen) in
the other is analyzed toshow that an a priori characteristic of
this portion of the viralgenome is sufficient retention of original
internal repetitious-ness.
Odds Against the Coding Sequence Having a LongAlternative Open
Reading Frame
The complete genomic DNA base sequences of human hepa-titis B
viruses of subtypes ayw (3182 base pairs long), adw(3200 base pairs
long), and adr (3188 base pairs long) weredetermined by Galibert et
al. (7) and by Ono et al. (8).Shown in Fig. 1 is the 2519-base-long
segment of one DNAstrand of subtype ayw (7) that encodes an 832
amino acidresidue long protein suspected of being either DNA
polymer-ase (8-10) or reverse transcriptase (11-14). Thus, the
aminoacid sequence shown below the base sequence of Fig. 1
isidentified as putative nucleic acid polymerase (Pol). It hasbeen
determined that the alternative reading frame of the
Abbreviations: Pol, putative nucleic acid polymerase; HBS
antigen,human hepatitis B surface antigen; RSV, Rous sarcoma virus;
bp,base pair(s).
3781
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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3782 Evolution: Ohno Proc. Nati. Acad2 Sci. USA 81 (1984)
base sequence specifying amino acid residues 334-570 of Pol the
coding sequence extremely rich in G+C has a betteralso encodes HBS
surface antigen of hepatitis B Dane parti- probability of having a
long alternative open reading frame.dles (7, 8, 15). In Fig. 1, the
226 amino acid residue long However, the AT/GC ratio of the base
sequence shown insequence of HBS antigen is shown above the base
sequence Fig. 1 was found to be 1.03/1.00. The 228 codons
specifyingidentified as HBS (fourth row from the bottom of Fig. la
to amino acid residues 343-570 of Pol (fourth row from the
bot-eighth row of Fig. lb). Inasmuch as the AT/GC ratio of three
tom of Fig. la to eighth row of Fig. lb) included 27
codonschain-terminating base triplets TGA, TAG, and TAA is 7/2,
that ended in TG (-TG), 6 codons that ended in TA (-TA), 48
aA TG CC C CT A TC CTATCA ACAC TTCC G GAGA CTACTG T T G TTAGACG
A CGAGG C A GG T CC CPOL 1 MET PRO LEU SER TYR GLN HIS PHE ARG ARG
LEU LEU LEU LEU ASP ASP GLU ALA GLY PRO
C TAGAG GA A GAACT CC CT CG C CTC G CAGACG AAGC T CTCAATC G CCGC
GT CGCAGAAGATPOL 21 LEU GLU GLU GLU LEU PRO ARG LEU ALA ASP GLU ALA
LEU ASN ARG ARG VAL ALA GLU ASP
C T CAAT CTCG GGAA T CTCAA TG TTAG TAT TCCT TG G CA TCA TA A
GGTGG G GAACT T TACTPOL 141 LEU ASN LEU GLY ASN LEU ASN VALNW~ ILE
PRO TRP HIS HISS VAL GLY ASN PHE THR
POL 61 GLY LEU TYR SER SER THR VAL PRO VAL PHEASN PRO HIS TRP
LYS THR PRO SER PHE PRO- I~~M
A A TATACAT T TACACCA AGACATTA T CAAAAAA TG TGAACAGT T T GTAGG
CC CAC T CACA
POL 8l jrJ ILE HIS LEU HIS GLN ASP ILE I LE LYS LYS CYS U GLN
PHE VAL GLY PRO LEU THRG TTAATGAGAAAAGA AGATT G CAAT TGATT A TG CC
T CCCAGG T T TTAT C CAAAGG T TAC C
POL 101 VALMIJ'~, U LYS ARG ARG LEU GIN LEU I LE MET PRO PRO ARG
PHE TYR PRO LYS VAL. THR
POL 121 Lys TYR LEU PRO LEU ASP S GLY ILE YS PRO TYR TYR PRO GLU
HIS LEU VAL1j1 1 HIS
POL 1141 TYR PHE GIN THIj HS YR LE HI TR LU TP LY ALA GLY lILE
LEU TYRj~S ARG
POL 161 GLU THR THR HISEWR ALA SER PHE CYS GLY SER PRO TYR SER
TRP GLU GIN ASP LEU GIN
HBs-163 MET SLY GLN A'SN LEL 'SLR THK SLR ASN 'RO LII GLY 'lIE
PH5 PRO ASP HIS GLN LEU ASP
POL 181 HIS GLY ALA GLU SER PHE HIS GIN GIN SER SER GLY ILE LEU
SER ARG PRO PRO VAL GLY
ItBS-143 PRO ALA PHlE AR(; ALA ASN SIHR ALA ASN PRO ASP TRI' ASP
PHE ASN PRO ASS LYS ASP THRT C CAGC CT T CAGA G CAA ACA C CG CAAAT
C CAGAT T G GGACT T CAA TC C CAACAAG GACA C
POL 201 SER SER LEU GIN SER LYS HIS ARG LYS SER ARO LEU GLY LEU
GIN SER GIN GIN GLY HIS
HJBS-123 TRP PRO ASP ALA ASN LYS VAL I;LY ALA IL) A1.LAPiL. (;LY
L.L GSLY Pli I. TSR PRO PRO HIS
POL 22 LEU ALA ARG ARG GIN GIN GLY0j3 SER TRP SER ILE ARG ALA
GLY PHE HIS PRO THR ALA
HBS-103 GLY GLY LEU LEU IL) rRI' SLR PRO I;N AlA IN (I1.) ILL
LII' GLN TSR ILL PRO ALA ASN
POL 2141 ARG ARG PRO PHE GLY VAL GLU PRO SER GLY SER GLY HIS THR
THR ASN PHE ALA SER LYS
KBS -83 PRO PRO PRO ALA SIR [SHR AsS ARG GLN SI R (LS ARI USLY
RI) rtSR PRO LLL ',LR PRO PRO
POL 261 SER ALA SER CYS LEO HIS GIN SER PRO VAL ARG LYS ALA ALA
TYR PRO ALA VAL SER THR
IJE -63 LEU AR(; ASN SIR HIS PRO 11.LN ALA MI.T (LN RI'K1
A.5> S1' IR15 'AiR PHlL 115 OLN STR LLL
POL 281 PH'in J LYS HIS SER SER SER GLY HIS ALA VAL GLU PHE HIS
ASN LEO PRO PRO ASN SER
H~s -143 GLN) ASP' 'RO SRI; VAL ARG IL) LEL' [YR PHII IRS ALA
(LI ILS SLFR SLR SER GLY TIR SAL
POL 301 ALA ARG SER GIN SERO J ARG PRO VAL PHE PRO CYS TRP TRP
LEU GIN PHE ARG ASN SERS
HBS -23 AS?) PRO SAL LiLU PIE iIR ALA SIFR IRo III >10K 'AR
ILl. 1II "SL AR(G ILL GLY AS)' PRO
POL 321 PRO CYS SER P TYR CYS LEU SER LEO ILE VAL ASN LEO LEO
GLU ASP TRP GLY PRO
H~s -3 ALA LI L ASN MET GLU ASN ILE THR SER GLY PHE LEU GLY PRO
LEU LEO VAL LEO GIN ALAT GC G CT GA ACATGGAGA ACAT CACA TCAG GA TT
C CTA GGACC C CTT CT C GT G TTACAGG C
POL 3141 cys 4ALAW HIS GLY GLU HIS HIS ILE ARG ILE PR01Ga THR
PRO SER ARG VAL THR GLY
HEs 18 GLY PHE PHE LEU LEU THRI ARG ILE LEU TieR I LE PRO GIN
SER LEU ASP SER TRP TRP THRGG G GT T TT TC TT GT TGACA AGAAT C CT
CACA A TACC G CAGA GT CT AGACT C GT G GTGGA C
POL 361 GLY VAL PHE LEU VALW j' LYS ASN PRO HIS ASN THR ALA GLU
SER111G LEO VAL VAL ASP
'lBs 38 SER LEU ASN PHE LEU GLY GLY THR THR VAL CYS LEO GLY GIN
ASN SER GIN SER PRO THRT T CT C TCAATT T TCTAGGG G GA A CTAC C GT
GT GT CTTGGCCAAAA TT CGCAGT CC CCAA C
POL 381 PHE SER GIN PHE SERing GLY ASN TYR ARG VAL SER TRP PRO
LYS PHE ALA VAL PRO AS1)
HBs 58 SER ASN HIS SER PRO THR SER CYS PRO PRO THR CYS PRO GLY
TYR ARG TRP MET CYS LEOC TC CAA T CAC T CA CCAACC T CT T GTC C
TCCAACT TGTC CT G GT TAT C GC TGGA T GTGTC T
POL 1401 LEU GIN SER LEU THR ASN LEU LEU SER SER ASN LEU SER TRP
LEO SER LEU ASP VAL SER
FIG. 1. The 2519-base-long cDNA sequence of human hepatitis B
subtype ayw genome that encodes the 832 amino acid residue long Pol
inone reading frame and the 226 amino acid residue long HBS antigen
in the other (7). This corresponds to =80% of the one genomic DNA
strand.The Pol coding sequence is identified by the amino acid
residues that it specifies placed immediately below the rows of
base sequences, whereasthe sequence for HBS antigen is identified
by the corresponding amino acid residues placed immediately above
the rows of base sequence.Amino acid residues at both ends of each
row are numbered. In the case of the ayw subtype, HBS antigen
coding sequence is preceded in its 5'end by an additional
163-codon-long potential coding sequence that begins with a chain
initiator ATG (extreme left of row 10 in a). Amino acid
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Evolution: Ohno Proc. Natl. Acad. Sci. USA 81 (1984) 3783
codons that started with A (A--), and 54 codons that started
which becomes (0.9633)226. Thus, the random probability ofwith G
(G--). Thus, the random probability of the above cod- the
above-noted Pol coding segment also encoding the 226ing segment
having the alternative reading frame open for amino acid residue
long HBS antigen by its one particular226 amino acid residues ofHBS
antigen can be calculated as: alternative reading frame becomes
2.15 x 10-4. This revela-
/2748 226 tion makes us realize a peculiarity in the original
construc-1i- ~-2-x7 _4 + (6 102 tion of the above-noted Pol coding
segment in that, although
228( 28 2) 28 228 there was no shortage of codons ending in TG
(-TG), suchb HBs 7° ARG ARG PHE I LE I LE PHE LEU PHE I LE LEU LEU
LEU CYS LEU I LE PHE LEU LEU VAL LEU
G C G G C G T T T T A T C A T C T T C C T C T T C A T C C T G C
TG C T A TG C C T C A T C T T C T T G T T G G T T C TPOL421 ALA ALA
PHE TYR HIS LEU PRO LEU HIS PRO ALA ALA MET PRO HIS LEU LEU VAL GLY
SER
HBS 98 LEU ASP TYR GLN GLY MET LEU PRO VAL CYS PRO LEU ILE PRO
GLY SER SER THR THR SERT C T G G A C T A T C A A G G T A T G T T G
C C C G T T T G T C C T C T A A T T C C A G G A T C C T C A A C A A
C C A G
PoL 441 SER GLY LEU SER ARG TYR VAL ALA ARG LEU SER SERN&
SER ARG ILE LEU ASN ASN GLN
HBS 118 THR GLY PRO CYS ARG THR CYS MET THR THR ALA GLN GLY THR
SER MET TYR PRO SER CYSC A C G G G A C C A T G C C G G A C C T G C
A T G A C T A C T G C T C A A G G A A C C T C T A T G T A T C C C T
C C T G
POL 461 HIS GLY THR MET PRO ASP LEU HIS TPYR CYS SER ARG ASN LEU
TYR VAL SER LEU LEU
BS 138 CYS CYS THR LYS PRO SER ASP GLY ASN CYS THR CYS I LE PRO
I LE PRO SER SER TRP ALAT T G C T G T A C C A A A C C T T C G G A C
G G A A A T T G C A C C T G T A T T C C C A T C C C A T C A T C C T
G G G C
POL 481 LEU LEU TYR GLN THR PHE GLY ARG LYS LEU HIS LEU TYR SER
HIS PRO ILE ILE LEU GLY
HBs 158 PHE GLY LYS PHE LEU TRP GLU TRP ALA SER ALA ARG PHE SER
TRP LEU SER LEU LEU VALPL5 T T T C G G A A A ATTC C T AT G G G A G
T G G G C C T C A G C C C G T T TC T C C T GGC T C AG T TTA
CTAGTPOt. 501PHE ARG LYS I LE PRO MET GLY VAL GLY LEU SER PRO PHE
LEU LEU ALA GLN PHETHFGTCA
HBS 178 PRO PHE VAL GLN TRP PHE VAL GLY LEU SER PRO THR VAL TRP
LEU SER VAL I LE TRP METG C C A T T T G T T C A G T G G T T C G T A
G G G C T T T C C C C C A C T G T T T G G C T T T C A G T T A T A T
G G A T
POL 521 ALA ILE CYS SER VAL VAL ARGO1 ALA PHE PRO HIS CYS LEU
ALA PHE SER TYR MET ASPIHBS 198 MET TRP TYR TRP GLY PRO SER LEU TYR
SER ILE LEU SER PRO PHE LEU PRO LEU LEU PRO
G A T G T G G T A T T G G G G G C C A A G T C T G T A C A G C A
T C T T G A G T C C C T T T T T A C C G C T G T T A C CPOL 541 VAL
VAL LEU GLY ALA LVS SER VAL GLN HIS LEL_ U SER LEU PHE THR ALA VAL
THR
HES 218 ILE PHE PHE CYS LEU TRP VAL TYR ILEAA T T T T C T T T T
G T C T T T G G G T A T A C AT T T AAA C C C T AA C A A A A C AA A
G AG A T G G G G T T A C
POL 561 ASN PHE LEU LEU SER LEU GLY I LE HIS LEU ASN PROMW LYS
THR LYS ARG TRP GLY TYR
T C T C T A A A T T T T A T G G G T T A T G T C A T T G G A T G
T T A T G G G T C C T T G C C A C A A G A A C A C A T CPOL 581 SER
LEU ASN PHE MET GLY TYR VAL ILE GLY CYS TYR GLY SER LEU PRO GLN GLU
HIS ILE
A T A C A A A A A A T C A A A G A A T G T T T T A G A A A A C T
T C C T A T T A A C A G G C C T A T T G A T T G G A A APOL 601 ILE
GLN LYS ILE LYS GLU CYS PHEEM LYS LEU PRO ILEj~a ARG PRO ILE _W TRP
LYS
G T A T G T C A A C G A A T T G T G G G T C T T T T G G G T T T
T G C T G C C C C T T T T A C A C A A T G T G G T T A TPOL 621 VAL
CYS GLN ARG ILE VAL GLY LEU LEU GLY PHE ALA ALA PRO PHE THR GLN CYS
GLY TYR
C C T G C G T T G A T G C C T T T G T A T G C A T G T A T T C A
A T C T A A G C A G G C T T T C A C T T T C T C G C C APOL 641 PRO
ALA LEU MET PRO LEU TYR ALA CYS I LE GLN SEES GLN ALA PHE THR PHE
SER PRO
A C T T A C A A G G C C T T T C T G T G T A A A C A A T A C C T
G A A C C T T T A C C C C G T T G C C C G G C A A C G GPOL 661 THR
TYR LYS ALA PHE LEU CYSS GLN TYR LEU ASN LEU TYR PRO VAL ALA ARG
GLN ARG
C C A G G T C T G T G C C A A G T G T T T G C T G A C G C A A C
C C C C A C T G G C T G G G G C T T G G T C A T G G G CPOL 681 PRO
GLY LEU CYS GLN VAL PHE ALA/_ ALA THR PRO THR GLY TRP GLY LEU VAL
MET GLY
C A T C A G C G C A T G C G T G G A A C C T T T T C G G C T C C
T C T G C C G A T C C A T A C T G C G G A A C T C C T APoL 701 HIS
GLN ARG MET ARG GLY THR PHE SER ALA PRO LEU PRO ILE HIS THR ALA GLU
LEU LEU
IsG C A G C T T G T T T T G C T C G C A G C A G G T C T G G A G
C A A A C A T T A T C G G G A C T G A T A A C T C T G T T
POL 721 ALA ALA CYS PHE ALA ARG SER ARG SER GLY ALA ASN ILE I LE
GLY THR_ FV SER VAL
G TAAACACTGGATCPOt. 741 VAL LEU SER ARG LYS TYR THR SER PHE PRO
TRP LEU LEU GLY CYS ALA ALA ASN TRP ILE
C T G C G C G G G A C G T C C T T T G T T T A C G T C C C G T C
G G C C C T G A A T C C T G C G G A C G A C C C T T C TPOL 761 LEU
ARG GLY THR SER PHE VAL TYR VAL PRO SER ALA LEU ASN PRO ALA ASP ASP
PRO SER
C G G G G T C G C T T G G G A C T C T C T C G T C C C C T T C T
C C G T C T G C C G T T C C G A C C G A C C A C G G G GPOL 781 ARG
GLY ARG LEU GLY LEU SER ARG PRO LEU LEU ARG LEU PRO PHE ARG PRO THR
THR GLY
C G C A C C T C T C T T T A C G C G G A C T C C C C G T C T G T
G C C T T C T C A T C T G C C G G A C C G T G T G C A CPOL 801 ARG
THR SER LEU TYR ALA ASP SER PRO SER VAL PRO SER HIS LEU PRO ASP ARG
VAL HIS
T T C G C T T C A C C T C T G C A C G T C G C A T G G A G A C C
A C C G TG A C G CC C A C C A A A T A T T G C C CPOL 821 PHE ALA
SER PRO LEU HIS VALAALA TRP ARGPRO PRO
residues specified by this additional coding sequence are shown
in smaller letters contiguous with HBS antigen and they are
numbered back-ward starting from HBS antigen -163 to HBS antigen
-1. All chain terminating base triplets are identified by solid
boxes placed in their properreading frames; those belonging to the
same reading frame as the HBS antigen coding sequence are placed
immediately above the base triplets,while a single chain terminator
of Pol coding sequence is placed immediately below TGA (bottom row
in b). The rest are placed below the Polamino acid sequence, as
they belong to the third unused reading frame.
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Proc. NatL. Acad Sci. USA 81 (1984)
codons are not followed by those starting with A (A--), andthat
codons starting with either A or G, (A--) and (G--), arenot
preceded by those ending in TA (-TA). Such avoidanceof particular
codon combinations by the Pol coding segmentmust have been inherent
in the original construction of hu-man hepatitis B viral genome,
for if it had not preexisted,natural selection has no means at its
disposal to favor thesubsequent creation of the alternative open
reading frame forHBS antigen by the Pol coding segment. All that
natural se-lection has been doing is maintaining the functional
integrityof the preexisting HBS antigen coding sequence
togetherwith that of the overlapping Pol coding sequence.
Internal Repetitiousness Embodied in the Base SequenceThat
Specified Pol and HBS Antigen in DifferentReading Frames
The tendency of one of the two alternative reading frames ofthe
2496-base-long Pol coding sequences shown in Fig. 1 toremain open
indeed appears as an inherent and original char-acteristic of this
particular base sequence, for the first alter-native reading frame
that in part encodes the 226 amino acidresidue long HBS antigen
contains altogether only 12 chain-terminating codons per 2496
bases, each identified in Fig. 1by a solid box placed above the
base sequence. In sharp con-trast, the second alternative reading
frame contains as manyas 38 chain terminators per the same number
of bases, eachidentified in Fig. 1 by a solid box placed below the
rows ofamino acid residues of Pol. Even if we exclude from
consid-eration the 684-base-long segment where the first
alternativereading frame encodes HBS antigen (fourth row from
thebottom of Fig. la to eighth row of Fig. lb) the difference
innumbers of chain terminators remains 11 chain terminatorsfor the
first alternative reading frame versus 28 chain termi-nators for
the second alternative reading frame. Indeed, inthe case of the
first alternative reading frame of ayw subtypeshown in Fig. 1, the
coding sequence for the 226 amino acidresidue long HBS antigen is
preceded on its 5' side by anadditional 618-base-long open reading
frame (Fig. la, rows7-18). Furthermore, due to the appropriate
appearance ofthe chain initiator ATG (Fig. la, row 10, far left), a
distal80% of this additional open reading frame has the potential
tospecify a 163 amino acid residue long polypeptide chain thatis
contiguous with HBS antigen.The inherent and original
characteristics of the base se-
quences shown in Fig. 1 that enable it to encode Pol andHBS
antigen in two different reading frames are apparentlyderived from
its ultimate ancestry, the ancestor being therepeat of a base
oligomer or oligomers. In Fig. 2, six separateinstances of still
discernible tandem oligomeric repeats aretabulated. These tandem
oligomeric repeats are accompa-nied by amino acid residues of HBS
antigen they specify fortheir easy identification in Fig. 1. Only
those found withinthe 684-base-long segment that encodes both Pol
and HBSantigen are tabulated (fourth row from the bottom of Fig.
lato eighth row of Fig. lb). In some instances, tandemly ar-ranged
copies are translated in the same reading frame.Thus, in HBS
antigen the tetrapeptidic sequence Ser-Pro-Thr-Ser recurs in rapid
succession, occupying amino acidposition 55-58 and then 61-64, as
shown at the top of Fig. 2.In other instances, two consecutive
copies of the base oligo-mer are translated in different reading
frames. Thus, twopentapeptidic sequences, Pro-Leu-Ile-Pro-Gly and
Ser-Ser-Thr-Thr-Ser, of amino acid positions 108-112 and then
113-117 of HBS antigen are encoded by two 16-base-long ho-mologous
base sequences tandemly arranged (73% base se-quence homology).
Overall, the entire base sequence of Fig.1 appears to have
descended from the nanodecameric re-peat, the oligomeric unit
sequence being something like
C-T-T-C-T-G-G-T-G-C-T-C-C-A-A-C-C-A-G, three consecutive
Hs (AYW) ANTIGEN 54 GSL SEN PRO T SER 58A G T CC C C A A C C
60 HIS SER PRO THR SER CYS PRO 66CACTCACCAACC TC TTG TCC T
6;7 PRO THt --- CYS PRO 70c.AACT - - - IT **
Hts(AYW) ANTIGEN ILE ILE P1H LEU P1H ILE 1EU LEU 88ATCATC TTCC
TCTTCATC C TGCTG
1 LU ILE P LEU LEU VAL .EU LEU 98' I . A. 1 C I I . T I G II G G
I T CI T C. I G
HS (AYW) ANTIGEN 108 PRO LEU ILE PRO SLYTCC TC TAAT TCCAGGA
113 SER SE R THR DHR SERI C. C.I fA
AC A A i i
A
HBs (Ayw) ANTIGEN 19 SLY PRO CYSG & *6A i A I 6i
122 ARG THR CYS METC G G A C C - T G C A T G
L30 SLY THN CYS METG G AA5CC G C ATG
* *- * ---
HBs (AYW) ANTIGEN
Has (AYW) ANTIGEN
165 TRP ALA SER ALA ARGG T G G G. CC T C A G C CC G
184 VAL SLY LEU SER PROG. GTAGSGG C T T TC C C C C
*-- *- *.-190 VAL TRP LEU SERG+ I T T j 6. C I .A
211 PRO PME LEUCCC TTTTTA
214 PRO LEU LEUCTC SC TC TI A
217 PRO ILE PEAA IIII C
112
121
125
133
169
188
193
213
216
219
FIG. 2. Six examples of oligomeric tandem repeats found
withinthe 684-base-long segment simultaneously encoding Pol and
HBSantigen (fourth row from bottom of Fig. la to eighth row of Fig.
lb)are tabulated here. In each example, one unmarked oligomeric
unitis considered as the prototype, and homologous bases in
othercopies are identified by asterisks. Each oligomeric unit is
accompa-nied by corresponding amino acid residues of HBS antigen.
Whenprototype and copies are translated in the same reading frame,
ho-mologous amino acid residues of copies are also identified by
aster-isks.
copies of it translated in three different reading frames
orig-inally giving the nanodecapeptidic
Leu-Leu-Val-Leu-Gln-Pro-Ala-Ser-Gly-Ala-Pro-Thr-Ser-Phe-Trp-Cys-Ser-Asn-Glnperiodicity
to all three polypeptide chains it encoded. Manytripeptidic
segments included in the above nanodecapeptidicunit occur at least
once either in a Pol amino acid sequenceor in an HBS antigen
sequence, and sometimes in both. Forexample, the Leu-Leu-Val
tripeptide occupies amino acidposition 94-96 of HBS antigen and
position 436-438 of Pol(first row of Fig. lb). Furthermore, the
Leu-Leu-Val-Leu-Gln pentapeptide is seen in position 12-16 of HBS
antigen(fourth row from the bottom of Fig. la). When the aminoacid
composition of 832 amino acid residue long Pol and 226amino acid
residue long HBS antigen of ayw subtype werecompared, the overall
similarity between the two was ratherstriking. Furthermore, this
similarity increased if the addi-tional open reading frame
immediately upstream of HBSantigen coding sequence was considered
as a part of HBSantigen coding sequence (Fig. la, HBS -163 to 226).
Itshould be pointed out here that the amino acid compositionof the
primordial nanodecapeptidic unit proposed above iscompatible with
the observed amino acid compositions ofboth Pol and HBS antigen.
For example, both Pol and HBSantigen were characterized by the
relative paucity of Glu,Lys, and Tyr, and these three residues are
not included inthe nanodecapeptidic unit. By contrast, usually
uncommonPhe, Cys, Trp were relatively abundant in both Pol and
HBSantigen (6.72%, 3.62%, and 4.13%, respectively, for Pol;7.08%,
6.19%, and 5.75%, respectively, for HBS antigen),
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Proc. Natl. Acad. Sci. USA 81 (1984) 3785
PbL (AY) 58 VAL GLY LEU SER PRO PE LEU LEU
51BGTGGGCCTCAGCCCGTTTC TCCTG
SS (ayw) 1 VAL GLY LEU SER PRO 188A G iIT T C iiiC
(AW) 2B LE sER PRo PHE Le PRo 214TIGtgTiiCIIITIAiCg
PR. (AW) 26 TYR CYS LEsu SER LEJ IEVLAL 32IA . T G ccIcTC C T A
T CPci (AvW) 412 LE SER 7P WL SER W ASP VAL 419
TTGTC CTGGTTATCGC TGGATGTG
H AS)ay) PM sPE W LEu sEE LEa L VAL I7II c I; . I G G. C A.C GT
T I A c T A T ~ F~V Pa.. .17 LEU-Hs-1YR4VT-ASP-ASP{-
--LEu-LEIJ-A.A-ALA-ALA-SER-s-HS-A-LY-LEU-GlU 195
N~s(AYw) 201 TRP GLY PRO SER LEU H POL 53 (5%) PHE-SE R-PE- - P
1 [L-L LYS-SR-VAL-GLN s-LE -GL 53 (5i)T G G G G G C C A A G T C T
G
POL (AYW)i78 TR GLYS' S E" 5 HB Pci 728
ARG-SER-GLY-ALAA-ILE-ILE-GLY-THR-P-ASP -SER- SAL-VAL-LD-LYS-1YE
746
I i i i i T T A C T C I C I A RW P1 68 UlY-Av.-PRD-LYS-
.A-IE-LYS-THR-ASP-SN-LY- s8v-vs-PHE-TM-SR-LYS-SER 701
FIG. 3. (Left) Three examples of segmental amino acid sequence
homology between Pol and HBS antigen are shown accompanied by
thecorresponding coding bases for each. (Right) Two segments of
human hepatitis B virus ayw subtype Pol (Hb Pol) amino acid
sequence thatshow reasonable homology with portions of two
suggested active sites of RSV Pol (Rous sarcoma virus reverse
transcriptase) are shown, eachaccompanied by a corresponding
segment of RSV Pol. Amino acid residues that are thought to remain
invariant in virus reverse transcriptasesof divergent oncogenic
retroviruses are shown in large capital letters (14, 16), as are
residues homologous with the above that are found in twosegments of
Hb Pol. The tripeptides, Val-Val-Leu, found in both segments of Hb
Pol are enclosed in a box.
and all three are represented once each in the nanodecapep-tidic
unit. On the other hand, the fact that the unused secondalternative
reading frame had a chain terminator recurring,on the average,
every 21.9 codons (38 chain terminators per832 codons) would have
been more readily explainable iffour base triplets in the proposed
primordial nanodecamericunit, TGG, TGC, CAA, and CAG, that can
easily change tochain terminators, TGA, TAA, and TAG, were arranged
inthe same reading frame.
Segmental Homology Between Pol and HBS Antigen and thePutative
Polymerase Active Sites of Pol
Inasmuch as the base sequence of Fig. 1 apparently
consistsmainly of variously degenerated copies of the
primordialoligomeric unit or units that are translated in various
readingframes within both Pol and HBS antigen-coding sequences,as a
corollary to the observed recurrence of various oligo-peptides in
each, segmental amino acid sequence homologyis expected between Pol
and HBS antigen. Three of the moreobvious examples of such
segmental amino acid sequencehomology between Pol and HBS antigen
are tabulated in Fig.3. At the top of Fig. 3, the octapeptidic
sequence occupyingposition 508-515 of Pol finds its homologues in
two segmentsof HBS antigen. The pentapeptidic portion
Val-Gly-Leu-Ser-Pro is also found in position 184-188 of HBS
antigen (Fig.lb, rows 5 and 6), whereas another overlapping
pentapepti-dic portion, Leu-Ser-Pro-Phe-Leu, is also found to
occupyposition 209-213 of HBS antigen (Fig. lb, rows 5 and 7).
Theother pentapeptide, Ser-Trp-Leu-Ser-Leu, also occurs onceeach in
Pol (position 413-417) and in HBS antigen (position171-175) as
shown in the middle of Fig. 3.Recently, the amino acid sequence
homology between the
certain carboxyl-terminal region of human hepatitis B Poland the
certain amino-terminal region of Pol (reverse tran-scriptase) of
Rous sarcoma virus (RSV) and other oncogenicretroviruses was
revealed (14). More precisely, amino acidresidues 499-596 of human
hepatitis B Pol (Fig. lb, rows 4-9) were found to be 22% homologous
with residues 141-237ofRSV Pol. It is of interest to note that the
octapeptidic Val-Gly-Leu-Ser-Pro-Phe-Leu-Leu of Pol that finds its
homo-logues in HBS antigen is included in this putative active
sitesegment of Pol (Fig. 3 Left). The conserved core of this
puta-tive active site ofhuman hepatitis virus B Pol is
Phe-Ser-Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala-Lys-Ser of
position536-548, the corresponding sequence in RSV Pol being
Leu-His-Tyr-Met-Asp-Asp-Leu-Leu-Leu-Ala-Ala-Ala-Ser, asshown (Fig.
3 Right). On the other hand, more extensivecomparison between
various Pols of diverse oncogenic ret-
roviruses led another group of investigators (16) to place
themost evolutionary conserved putative active site not on
theamino-terminal region but on the carboxyl-terminal region ofthe
reverse transcriptase. The conserved core of this alterna-tive
active site in the case of RSV Pol was
Ala-Ile-Lys-Thr-Asp-Asn-Gly-Ser-Cys-Phe-Thr-Ser-Lys-Ser, occupying
po-sition 689-702 as shown (Fig. 3 Right). Interestingly,
furthertoward the carboxyl terminus of human hepatitis B virus
Pol,we also find the segment (position 733-746) with the se-quence
Ile-Ile-Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg-Lys-Tyr (Fig. 3
Right; Fig. lb, sixth and fifth rows from thebottom). The homology
between these two segments ofRSVPol and human hepatitis B virus Pol
is 35.7%. Not surprising-ly, the two putative active sites within
human hepatitis Polare also somewhat homologous with each other,
sharing theVal-Val-Leu tripeptide, thus again attesting to the
inherentand original internal repetitiousness of human hepatitis B
vi-rus Pol coding sequence.
This work was supported by the Jeff, Fred, and Ritchie
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