Nucleotide sequence and transcription of a human glycine tRNAGCC gene and nearby pseudogene

155

Gene, 43 (1986) 155-167 Elsevier GENE

1600

Nucleotide sequence and transcription of a human glycine tRNAGcc gene and nearby pseudogene (Recombinant DNA; restriction enzyme mapping; RNA polymerase III; promoter pBR322; J Charon 4A; RNA fingerprinting; RNA processing; Ah-like element)

mutation;

plasmid

Irma L. Pirtle, Randall D. Shortridge and Robert M. Pirtle * Departments of Chemistry and Biochemistry, North Texas State University/Texas 76203 (U.S.A.) Tel. (817)565-3537 (Received

January

21st, 1986)

(Accepted

March

lOth, 1986)

College of Osteopathic Medicine, Denton, TX

SUMMARY

A bacteriophage ,? clone containing a 15.4-kb human DNA fragment was isolated and found to contain a glycine tRNA gene and, 758 bp away, a pseudogene, both with an anticodon of GCC. The nucleotide (nt) sequence of a 1362-bp segment of this clone, encompassing the gene, pseudogene, and their flanking regions, was determined. The gene and pseudogene have an identical sequence of eight nt (5’-CAGCTGGA-3’) in their 5’-flanking regions immediately preceding the coding regions, as well as characteristic transcription termination sites of five consecutive T nt in the 3’-flanking regions. Neither of these genes has intervening sequences. Only one of the two genes was efficiently transcribed in vitro by RNA polymerase III in a HeLa cell-free system. During the course of transcription, primary transcripts of one gene were processed to yield mature-sized products. In contrast, the level of transcription of the second gene was significantly less than that of the first, and no mature-sized products could be detected. The nt sequence of the inefficiently transcribed gene has two base substitutions compared to the sequence of the efficiently transcribed gene, and the DNA sequence predicted from the human placental tRNA$, sequence. One of these nt substitutions is a C to T transition in the TTCG sequence within the B block of the characteristic internal split promoter sequence. The precursor-product relationships of the tRNA transcripts were established by comparing the RNase T, and RNase A fingerprints of the precursors and products.

INTRODUCTION

There are around 1300 human tRNA genes (Hatlen and Attardi, 1971), encoding 60-90 different isoaccepting species of tRNA (Lin and Agris, * To whom

correspondence

and

reprint

requests

should

be

addressed. Abbreviations: nucleotide(s);

bp, base pair(s); Pu, purine;

kb, kilobases

Py, pyrimidine;

SDS,

or 1000 bp; nt, sodium dodecyl

sulfate.

0378-l 119/86/%03.50 0 1986 Elsevier

Science Publishers

B.V. (Biomedical

1980). Despite the availability of modem recombinant DNA techniques, relatively few of these human tRNA genes or other mammalian tRNA genes have been isolated and sequenced (Sprinzl et al., 1985a). Some of these mammalian tRNA genes occur individually and are widely dispersed throughout the genome (Santos and Zasloff, 1981), whereas others are found in small clusters (Roy et al., 1982). However, it is not as yet clear how tRNA gene families are organized in the mammalian genome. Division)

156

In a previous paper, we described the structure and transcription of a tRNA gene encoding a tRNA& (Shortridge et al., 1985). Since it is important to obtain further information on mammalian tRNA gene structure and expression, we have continued with our studies of the human glycine tRNA gene family by determining the nucleotide sequence of a tRNAGiy GCC gene and nearby pseudogene, and studying the relative efficiencies of transcription of the two genes.

MATERIALS

AND METHODS

(a) Materials

The HeLa cell in vitro transcription system and [c~-~~P]GTP were obtained from New England Nuclear. Agarose (LE, NuSieve and SeaPlaque) was purchased from FMC Corp. RNase A was obtained from Cooper BioMedical, and RNase T, was supplied by CalBiochem. The [y-32P]ATP and [5’-32P]pCp for labeling DNA and RNA, respectively, were purchased from ICN Radiochemicals. Most reagents for synthesizing DNA were supplied by Biosearch, Inc. Methylene chloride and acetonitrile were purchased from Burdick and Jackson. The sources for other materials have been previously listed (Pit-tie et al., 1980a,b; Shortridge et al., 1985). (b) Isolation and restriction mapping of the genes

Human DNA fragments containing glycine tRNA genes were isolated by screening a human gene library contained in bacteriophage 1 Charon 4A (Slightom et al., 1980). The 2 phage clones containing these genes were grown on E. co/iK-12 DP50 supF in 2 x NZCYM-DT medium (Zehnbauer and Blattner, 1982) and plaque-purified (Benton and Davis, 1977; Zehnbauer and Blattner, 1982) using a hyb~dization solution consisting of 50 % formamide, 150 mM NaCl, 15 mM Na, . citrate, pH 7.0, 0.1% SDS, 1 mM EDTA, yeast carrier tRNA (10 hg/ml) and bovine liver [ 3’-32P]tRNA$-&, at 43°C (Heckman and RajBhandary, 1979). The phage DNA was purified by extraction with phenol and chloroform : n-octanol(24 : 1) and precipitated with ethanol. A 4.3-kb BamHI fragment of ;IhGly3 which was subcloned hybridized to 32P-labeled tRNA$,

into the BamHI site of pBR322. Plasmid DNA from the subclone, designated phGly3, was purified as previously described (Shortridge et al., 1985). The human DNA inserts from AhGly3 and phGly3 were characterized by restriction enzyme mapping using standard methods (Southern, 1979; Lawn et al., 1978) for aligning fragments derived from single and double enzymatic digestions of the cloned DNA. In addition, DNA fragments excised from 0.7% low-melting temperature SeaPlaque agarose gels were re-digested and fractionated on either 2% LE agarose or 4% NuSieve agarose gels as described by Parker and Seed (1980). Purified bovine liver [3’-“‘P]tRNA$& and unfractionated [3’-32P]tRNA were used as hybridization probes to localize tRNA genes. The 32P-labeling and sequence analysis of bovine liver tRNAg& have been previously described (Shortridge et al., 1985). Putative A/u-like elements were localized by Southern blot hybridization to restriction enzyme digests of hhGly3 using two 5’-32P-labeled synthetic oligodeoxyribonucleotides (5’-TCCCAGAATTTT GGGA-3’ and 5’-GGAGGCTGAGGCAG-3’). corresponding to two different regions of the consensus sequence of A&-like elements (Schmid and Jelinek, 1982) as hybridization probes. The hybridization conditions used were similar to those described by Szostak et al. (1979). The probes were synthesized using a Biosearch Sam One DNA Synthesizer by a modified phosphotriester procedure. The oligomers were purified by fractionation on a 20% denaturing polyacrylamide gel, and the nt sequence was verified by mobility-shift analysis (Wu et al., 1984).

RESULTS

(a) Isolation of the phage I clone

A E, clone designated IhGly3 was selected and plaque-purified for further study since it gave a strong positive signal upon autoradiography, when bovine liver [3’-32P]tRNA$c was utilized as hyb~dization probe during the screening of a human DNA library. The DNA of IhGly3 was characterized using restriction mapping and Southern blot hybridizations (Southern, 1979). It became apparent

157

(a)

(b) Hha

EGO RI

Barn HI

I

,

Hha

I

BarnHI

-

0.8 Kb

Fig. 1. Hybridization bromide-stained

of 32P-labeled

1% agarose

restriction

enzyme

indicated

radiogram

of a Southern

glycine tRNA,,,

gels (let? lanes)

to Southern

and corresponding

blots containing autoradiograms

in the figure. Sizes (in kb) of the DNA fragments

blot made from a 2% agarose

DNA. The scale to the right was derived

gel used to fractionate

digests

which hybridized double

from the sizes (in kb) of DNA fragments

that either LhGly3 contained at least two glycine tRNA genes, or alternatively, a single glycine tRNA gene with a large intervening sequence, since more than one fragment from a number of different restriction endonuclease digestions hybridized to the 32Plabeled tRNA probe (see Fig. 1). A 4.3-kb BumHI fragment (Fig. la) was subsequently isolated from AhGly3 and subcloned into the BamHI site of pBR322.

restriction

of IhGly3

(right lanes) that resulted restriction

to the tRNA endonuclease

DNA. (a) Ethidium

from cleavage are indicated. digestions

with the (b) Autoof IhGly3

from a Hind111 digest of i, DNA.

(b) Restriction enzyme mapping The size of the human DNA insert of LhGly3 was estimated to be 15.4 kb in length, based on the sum of the EcoRI fragments (Fig. la). Southern blot data indicated that only a single HhaI fragment of IlhGly3 (encompassed within a 4.3-kb BamHI fragment) hybridized to [3’-32P]tRNA$C (Fig. la). Other Southern blot data, as well as preliminary nt

158

sequence analyses, confirmed the existence of two different tRNA GcC genes, contiguous on the same DNA strand and separated by about 0.8 kb. The nt sequence of the coding regidns of the two tRNA genes revealed internal EcoRI and HinfI cleavage sites 21 and 16 bp from the 5’ end and 3’ end, respectively, of the structural genes. The glycine tRNA probe hybridized to the 0.8-kb and 4.3-kb EcoRI fragments (Fig. la) containing the intergenic region and the 3’ flank of the second gene, respectively. A 3.7-kb EcoRI fragment, containing only 21 bp of the first gene and its 5’-flanking region, did not hybridize under our hybridization conditions. Similarly, no hyb~dization was observed for Hinfi fragments containing only 16 bp of the second gene (Fig. lb). Double digestions of IhGly3 DNA with

(a)

BomH I Xho I

EcoR I BomH I Bamti I

EcoR I EcoR I

I

,’

c

/’

/’

EcoR I

EcoR I

Bgl II

/’

/ /’

/ I

AJ

---__

-_ -...

--__

--__

--._

--__

--._ --__

--__

--__

/

I

Hinf I

Sph I

I

I Dde I

Sph I

I

I

thnf I

Ava I Hind III

Sau3A I

Xba I

I

I

I Ava I

Aha III

BstN I Rso I *

*

*

t-“---W

*

*

.

. .

-

*

. -

.

I

Hha I

w -

‘?

I us0 I

Avo I

--_ c

Fnu4H I

Hmf I

Hpo I

EcoR I

Sou3AI

EcoR I

Bgl II

Bgl II Xbo I Sst I Xbo I

11

Bgl

(b)

EcoRI + HhaI or EcoRI + BamHI revealed that there was a HhaI cleavage site about 0.2 kb and a BarnHI cleavage site about 1.6 kb from the 3’ end of the second gene. There are no PvuI, KpnI or Sal1 cleavage sites within the human insert of IhGly3. These and other restriction mapping data are summarized in the restriction map of LhGly3 in Fig. 2a and the sequencing strategy shown in Fig. 2b. Southern blot analysis of IhGly3 DNA hyb~diz~ with unfractionated bovine liver [ 3’-32P]tRNA probe yielded data identical to those obtained using as probe, indicating that there were only tRNA& probably no additional tRNA genes in IhGly3 DNA. ~yb~d~ation of synthetic &-element probes to Southern blots of dhGly3 DNA demonstrated that there were at least three putative A/u-like elements on the 3’ side of gene II in the human DNA

.

. *

Fig. 2. Restriction mapping and sequencing strategy, (a) Restriction map of a cloned 15.4-kb segment of human DNA, designated IhGly3, encompassing two glycine tRNAocc genes. The line represents the human DNA strand noncoding for tRNA. The hatched areas represent the 1 Charon 4A vector arms. The black rectangles (not drawn to scale) represent the structural genes. The gene on the left (5’ side) has been designated as gene I and the gene on the right as gene II. The dashed lines indicate the region of IZhGly3 DNA sequenced. (b) nt sequencing strategy. The restricted fragments of phGly3 were 5’-“P-labeled and sequenced in the direction and to the extent indicated by the arrows. The thick lines represent the structural genes. Fig. 3. Nucleotide sequence of a 1362-bp segment of the human DNA insert of phGly3 containing two glycine tRNA,,, genes. The b 3zP labeling and sequence analysis of DNA from phGly3 were done according to the procedures of Maxam and Gilbert (1980). Storage and analysis of the sequences of the DNA fragments were done using computer programs for the Apple IIe (Larson and Messing, 1983)

159 80

70 60 50 40 30 20 10 GATcTTTAAGGATAAcAGAGTAAGGTCTGGAACCTGCCCACAGGAGCCAAAAGAATTGCTGAGcATTcTcccATTGccAT CTAGAAATTCCTATTGTCTCATTCCAGACCTTGGACGGGTGTCCTCGGTTTTCTTAACGACTCGTAAGAGGGTAACGGTA 150 140 130 120 100 110 90 TCATGGACTTTTCTTTTGAACAGCTGAGCAGAATGTCAAGCATGCATGCCCCACCTCCTCCCATAGGTTTGGCTGGTAAA AGTACCTGAAAAGAAAACTTGTCGACTCGTCTTACAGTTCGTACGTACGGGGTGGAGGAGGGTATCCAAACCGACCATTT

160

230 210 220 200 190 170 lG0 ATCCAGCAGCTGTCCATGCTGGACCCATCTCAGGTGTCAGGTTACAGCTGTGCCATCACCACTGTGAATCAGAGCAACAA TAGGTCGTCGACAGGTACGACCTGGGTAGAGTCCACAGTCCAATGTCGACACGGTAGTGGTGACACTTAGTCTCGTTGTT

240

310 290 300 280 CATTGGTGGTTCAGTGGTAGAATTCTCGCCTGCCACGCGGGAGGCCC GTAACCACCAAGTCACCATCTTAAGAGCGGACGGTGCGCCCTCCGGG

270 260 250 AACAGCTGGAGGCAGAACAGCACTCAGCTGG TTGTCGACCTCCGTCTTGTCGTGAGTCGACC 330 x4o GGGTTCGATTCCCGGCCAATGC CCCAAGCTAAGGGCCGGTTACG

~

350 2 360 370 380 390 GCAGCTGAAAGCTTTTTGGCAGCTCTTGGAAAAAGAAAACTTGGAGAAATAAGTTAA CGTCGACTTTCGAAAAACCGTCGAGAACCTTTTTCTTTTGAACCTCTTTATTCAATT

320

400

470 440 450 460 410 420 430 CTTGGAGGGATAAGCTAGTGCGGGCCTTCAAAGGGAGGAGCTTTTTTTACTGGGAGAAACTAGAAGACTCGGGGATACAT GAACCTCCCTATTCGATCACGCCCGGAAGTTTCCCTCCTCGAAAAAAATGACCCTCTTTGATCTTCTGAGCCCCTATGTA

480

540 510 520 530 490 500 550 ATTTTGTGACCTCTCACTGAAATATGAGTGTTTGATTTTTGTTTTCTAATTCTAATTTTAGAAAATTTGGAAAGTAGAGA TAAAACACTGGAGAGTGACTTTATACTCACAAACTAAAAACAAAAGATTAAGATTAAAATCTTTTAAACCTTTCATCTCT

560

610 620 630 570 580 590 600 AAAATGCAAAGAAACAAGACTATCCCCAAGAGGCAATGAATGTTACAAATTTTGATGTATTTTTTCCAGTTTTTAATATA TTTTACGTTTCTTTGTTCTGATAGGGGTTCTCCGTTACTTACAATGTTTAAAACTACATAAAAAAGGTCAAAAATTATAT

440

650 680 690 700 710 640 670 TATACGTAGTTAAGATCATAGAACTTAACACAGCTTCACCTTCTGCTCTGTTTGTTTGAAGTTCACAGTGCCTCTTAATC ATATGCATCAATTCTAGTATCTTGAATTGTGTCGAAGTGGAAGACGAGACAAACAAACTTCAAGTGTCACGGAGAATTAG

720

730 740 750 760 770 780 790 CATAGTCAGAAAATAGAGTAACAGTTGCCTGGGTTTGAAGAAAAGGAGAATTGGGACTGACTGCTTACAGATGCCCGGTT GTATCAGTCTTTTATCTCATTGTCAACGGACCCAAACTTCTTTTCCTCTTAACCCTGACTGACGAATGTCTACGGGCCAA

000

810 020 030 040 850 860 870 TCTTTTTGGGGTTATGGAGATGTTCTAGAATTAAATAGAGGTGATGGGTGCACAACTTTGTGAATGTACTAAAAACTACC AGAAAAA~~~~AATA~~T~TA~AAGAT~TTAATTTAT~T~~A~TA~~~A~GTGTTGAAA~A~TTA~ATGATTTTTGATGG

GGO

090 900 910 920 930 940 950 TAGTTGAACACTTTGAAAATGGTGAAA~TTTTGTTATGTGAAATGTTATATGAATTATATCTCAATTTAAAAAAAAAAAA ATCAACTTGTGAAACTTTTACCACTTTAAAAACAATACACTTTACAATATACTTAATATAGAGTTAAATTTTTTTTTTTT

940

970 980 990 1000 1010 1020 1030 GTCAGTGCCTATTCCTCTAAATTAGAACCCAACAGCCCCTGCCACCTTTCTGCTTATGTGGCAAGAAGTCAAGCCCGCTA CAGTCACGGATAAGGAGATTTAATCTTGGGTTGTCGGGGACGGTGGAAAGACGAATACACCGTTCTTCAGTTCGGGCGAT 1050 1040 1070 1080 1090 GAAAGGAACCACTCCATCCTGTGGGTCGTGGCCTCAACTAAAAACATCCCTACCAGCTGG CTTTCCTTGGTGAGGTAGGACACCCAGCACCGGAGTTGATTTTTGTAGGGATGGTCGACC

1100

1040

1110 1120 CATTGGTGGTTCAGTGGT GTAACCACCAAGTCACCA

llE0 1190 1200 CCTATCTACCTTTTTAGATGGTTTTCC GGATAGATGGAAAAATCTACCAAAAGG

1130 1140 1150 1160 1170 AGAATTCTCGCCTGCCACGCGGGAGGCCCGGGTTTGATTCCCGGCCAGTGC TCTTAAGAGCGGACGGTGCGCCCTCCGGGCCCAAACTAAGGGCCGGTCACG

1210 1220 1230 1240 1250 1260 1270 TAAGCATTTTTTCACACAGCTGCTTGCCCAGGAGAGTATTACCCTAACCAGGATACATTCATGCCTCTTGGCTCTGGATT ATTCGTAAAAAAGTGTGTCGACGAACGGGTCCTCTCATAATGGGATTGGTCCTATGTAAGTACGGAGAACCGAGACCTAA

1280

1290 1300 1310 1320 1330 1340 1350 TTACTGCCTCATAAACAGCATTTCAGCAGATGAAAAGCTCACTTTGTACTAGTTCCCAGAGATGGTAAGAGCTAATTTGC AATGACGGAGTATTTGTCGTAAAGTCGTCTACTTTTCGAGTGAAACATGATCAAGGGTCTCTACCATTCTCGATTAAACG

1360

GC CG

and the IBM PC (Lagrimini et al., 1986) that (nt 273-343)

searches

et al., 1984) microcomputers. for the presence

of additional

and II (nt 1102-I 172). Transcription

Nucleotide tRNA

sequence

files were also analyzed

gene sequences.

The boxes

encompass

using a program glycine

(Shortridge

tRNA,,,

of both tRNA genes is from left to right (5’ to 3’) from the lower strand.

genes I

160

insert of ;IhGly3. These probes hybridized to a 0.9-kb SstI-BumHI fragment (Fig. 2a), near the center of the human DNA insert. The probes also hybridized to a 2-kb EcoRI-BglII fragment and a 0.8kb BglII-XbaI fragment derived from a region of AhGly3, toward the right arm of the /z Charon 4A DNA (Fig. 2a). (c) Nueleotide sequence The strategy used to sequence the two adjacent tRNA genes, and their flanking regions is shown in Fig. 2b. Overlapping fragments from both strands of this region of phGly3 were sequenced, ensuring the accuracy of the DNA sequence shown in Fig. 3. Neither of the two genes has introns nor encodes the 3’-terminal CCA sequence found in mature tRNAs. Both genes have an anticodon of GCC. Gene1 (nt 273-343 of Fig. 3) is identical to the nt sequence predicted from the RNA sequence of human tRNAsc (Gupta et al., 1979). The nt sequence of the noncoding strand of gene I is shown in the in Fig. 4. Gene II cloverleaf conformation (nt 1102-l 172 of Fig. 3) has two substitutions of nt 54 and 67 of mature tRNA (corresponding to nt 1155 and 1168, respectively, of Fig. 3), as indicated by the arrows in the cloverleaf structure shown

A G C70 C*G A*T T - A-G 51 A l

l

GaC65 T

:

A

GTG CTTG

G

E

8;;;

G T

A 20

TT

’ ‘G

G

C”c”C

;:

ddd 50

C 25T C-G T*G C G*C 3OC

AG

l

G40

l

G

c

c

T

A

Gc

G 45

AG 55

T

TC\

T

=

Fig. 4. Human glycine tRNA,,, gene I in the cloverleafconformation. The arrows indicate the two substitutions found in gene II when compared to human glycine tRNA,,, gene I.

in Fig. 4. In particular, the substitution of nt 54 does not conform to the highly conserved ‘ITCG sequence which corresponds to the T YCG sequence of mature tRNAs. There is little homology between the flanking regions of the two genes other than: (1) an identical sequence (S’-CAGCTGGA-3’) immediately preceding the coding region of the two genes (nt 265-272 and nt 1094-l 101, respectively, . . similar sequences ;ngi _C~~~~~AA~~~~~ nt 235-244, and 5’-CAACTAAAAACA->‘, nt 1075-1086} centered about - 32 and - 20, upstream from genes I and II, respectively; and (3) a stretch of five consecutive T nt in the 3’-flanking regions near the ends of both genes. The genes are separated by an intergenie 758-bp region. The intergenic region is A + Trich and has one block (nt 636-648) of 13 alternating Pu and Py bases. (d) In vitro transcription studies Both AhGly3 DNA and phGly3 DNA were successfully transcribed in a Heta cell in vitro transcription system (Manley et al., 1980) containing [c(-~*P]GTP, as shown in Fig. 5a. A major product with an electrophoretic mobility that corresponded was assumed to be the to that of [ 3 ‘-32P]tRNA$, mature-sized tRNA product, as shown in lanes 2-4. The products with electrophoretic mobilities slightly less than the bovine liver[ 3’-32P]tRNAb;& standard (Pirtle et al., 1980a) in lanes 2-4 of Fig. 5a, were assumed to be precursor tr~sc~pts of the glycine tRNA genes. These assumptions are supported by the fact that the 32P-labeled RNA transcription product hybridized to DNA fragments containing GCCgenes (not shown). From a comparithe tRNAG’y son of the electrophoretic mobilities of the putative precursor transcripts to the electrophoretic mobilities of the 3’-32P-labeled tRNA$& (75 nt) and the tRNA$ (86 nt) standards, it can be concluded that the putative precursor glycine tRNA transcripts are about 93-95 nt in length. The presence of at least one A&like element (S&mid and Jelinek, 1982) in phGly3 was indicated by the fact that a large transcript from a major band (indicated by A in Fig. 5a and also in a similar position in lane 1 of Fig. 5b) hybridized to Southern blots of BLUR8 DNA (Rubin et al., 1980). There was essentially no effect on the extent or degree of transcription when RNA

161

Gene it

Gene I 15

20

30

60

$0

125

16

20

30

60

90

Fig. 5. Fractionation of in vitro transcripts on 8% denaturing polyacrylamide gels. DNA from phGly3 and tlhGly3 was transcribed in a homologous HeLa cell in vitro transcription system (Manley et al., 1980) using reaction conditions reported by Shortridge et al. (1985). (a) Lane 1 contains 3’-“2P-labeled tRNAg& and tRNAIAG Lru from bovine liver as standards. Lanes 2-4 contain transcription products from 90 min incubations with: (2) dhGly3 DNA, (3) IhGly3 DNA + a-amanitin (2 pg/ml), and (4) phGly3 DNA. (b) Products derived from transcription of DNA fragments containing gene I and gene II. Lanes 1 and 2 contain transcription products derived from 90 min incubations with phGly3 DNA and a SphI digest of phGly3 DNA, respectively. The products derived from transcription of a 1.4-kb AhaIII fragment containing gene I during the indicated incubation periods (min) are shown to the left. The products derived from transcription of a 0.8-kb AhaIII-&I fragment containing gene II are shown to the right. Lane S contains [3’-3ZP]tRNA (unfractionated) from bovine liver as a standard.

polymerase II activity was inhibited by adding 01amanitin (2 pg/ml) to reaction mixtures containing either JhGly3 DNA (Fig. 5a, lane 3) or phGty3 DNA (not shown). The very large RNA transcripts toward the top of the gel (Fig. 5a) were not characterized. To assess the ability of each gene to direct transcription in the in vitro HeLa cell system, restriction fragments containing gene I and gene II were separated and transcribed individually. Using a 1.4-kb

AhaIII fragment containing gene I as a substrate for in vitro transcription (Fig. 5b), there was a progressive accumulation of a mature-sized product as the reaction progressed. Putative precursor tRNA transcripts started accumulating within the first 15 min after the reaction was started. After about 60 min incubation, little of the putative precursor and a preponder~ce of the mature-sized product was observed. The putative Mu-like element transcript was not observed in the transcription products from

162

?06 2

O7

t

*

2

L Fig. 6. Fingerprint

analyses

of the RNase T, and RNase A digestion

genes. The tRNA transcription

products

T, or RNase

by two-dimensional

A, and separated

of the mature-sized

transcription

product

from gene I. (c) RNase T, fingerprint

derived from gene II. Numbered

discussed

in RESULTS,

is electrophoresis

as reported

products

of tRNA transcripts

by Shortridge

et al. (1985)

electrophoresis-homochromatography

derived from gene I. (b) RNase A fingerprint transcript

circles indicate

section d. The oligonucleotides

derived

(Brownlee,

plate using homomixture

by position

C-30 (Brownlee,

digested

of the mature-sized

spots appearing

with either RNase

1972). (a) RNase T, fingerprint transcription

from gene II. (d) RNase A fingerprint

the loci of additional

were identified

derived from the glycine tRNA,oo

completely

product

in similar fingerprints

1972). The “B” represents

(not shown),

is homochromatography the location

derived

of precursor-sized

only (Pirtle et al., 1980b). The first dimension

on cellulose acetate at pH 3.5 for 40 min at 4800V. The second dimension

and 65°C on a DEAE-cellulose FF dye marker.

were isolated

of precursor-sized

transcript fingerprint

1

as

of each at pH 7.5

of the xylene cyan01

163

this fragment. In a separate experiment, a 0.8-kb AhaIII-SstI fragment containing gene II was tested as a substrate, and a si~~c~tly lower efficiency of transcription was observed relative to that shown for gene I (Fig. 5b). Although small quantities of the putative primary transcript of gene II accum~ated, no detectable amount of the mature-sized product could be detected. Transcripts migrating at a slower rate than the primary transcript from gene II were deduced to be related to transcription of a segment of the &-like element located between the SstI and BumHI sites in the 3’ flank of gene II (Fig. 2a). These tr~sc~pts appear to result from transcription of a 0.8-kb SstI-SphI fragment that co-purified with the AhaIII-SstI fragment containing gene II. This SstI-SphI segment is located immediately adjacent to the 3’-side of the~h~III-S~~I fragment containing gene II. In addition, a transcription product with the same electrophoretic mobility as these putative Mulike transcripts can be seen in lane 2 of Fig. 5b, which contains the transcription products of a SphI digest of phGly3. This would suggest the SphI cleavage site is within the DNA sequence of the putative Alu-like element, and that a segment of the A&-like element is located within the 0.8-kb SstI-SpkI fragment adjacent to gene II. The RNase T, and RNase A fingerprints of the 32P-labeled mature gene I transcript (Fig. 6,a and b, respectively) are consistent with the tDNA sequence of Fig. 4, and also with the degradation products predicted from human placental tRNA$& (Gupta et al., 1979). The RNase T, fingerprint of the supposed precursor tRNA derived from gene I has two spots not found in the RNase T, fingerprint of the mature tRNA transcript. These two spots would correspond to two unique oligonucleotides derived from the 3’-end of the precursor tRNA transcript, 5’-CAG-3’ (transcribed from nt 342-344 and nt 345-347 of Fig. 3, and shown schematically as spot 1 of Fig. 6a) and 5’-AAAG-3’ (transcribed from nt 351-354 of Fig. 3, and shown s~hematic~ly as spot 2 of Fig. 6a). The RNase A fingerprint of the putative precursor tRNA of gene I also contains a unique spot corresponding to 5’-GAAAGC-3’ (derived from nt 350-355 of Fig. 3, and shown schematically as spot 7 of Fig. 6b) which also originates from the 3’“flanking region of the precursor transcript. In addition, the RNase A fingerprints of both the putative gene I and gene II precursor transcripts

spot corresponding to contain a unique 5’-GGAGC-3’ (from nt 270-274 of gene I, shown schematic~y as spot 6 in Fig. 6b; and from nt 1099-l 103 of gene II, spot 10 of Fig. 6d), derived from the 5’-terminal regions of the precursor tRNAs of both genes. Thus, because the RNase T, and RNase A fingerprints of both the supposed precursor transcript and the tRNA product of gene I have similar fmgerprints, but yet have unique spots corresponding to oligonucleotides derived from the 5’and 3’-flanking regions of the precursor tRNA transcript as predicted from the DNA sequence, a precursor-product relationship between the transcripts of gene I can be strongly implied. The RNase T, and A fingerprints of the 32Plabeled transcript corresponding to the precursor tRNA of gene II (shown in Fig. 6,c and d, respectively) are consistent with the two base substitutions between genes I and II established by DNA sequence analysis. These variations would lead to two distinct differences between the RNaseT, Iingerprints and one difference between the RNase A fingerprints of the tRNA transcripts derived from genes I and II. The C54 to T54 positions change between genes I and II would result in the occurrence of the fragment 5’-UUCG-3’ in the RNase T, ~nge~~nt of the gene I transcript (which would correspond to spot 4 of Fig. 6a), whereas 5’-UUUG-3’ would occur in the RNase T, tingerprint of the gene II transcript (spot 9 of Fig. 6~). The A67 to G67 transitional change would result in the occurrence of the fragment 5’-CCAAUG-3’ in the RNase T, fingerprint of the gene I transcript (spot 3 of Fig. 6a), whereas T’XCAG-3’ would occur in the RNase T, fingerprint of the gene II transcript (spot 8 of Fig. 6~). In addition, the A67 to G67 transition would lead to the appearance of 5’-AAU-3’ in the RNase A ~nge~~nt of the gene I transcript (spot 5 of Fig. 6b), whereas this fragment would not appear in the RNase A fingerprint of the gene II transcript (Fig. 6d),

DiSCUSSION

The human glycine tRNAGcc gene I would encode a tRNA which is 100% homologous to human placental tRNAg& (Gupta et al., 1979), discount-

164

ing post-transcriptional modifications, Since tRNA$& gene I is accurately transcribed by RNA polymerase III in an in vitro HeLa cell tr~sc~ption system, it very likely represents a true structural gene for the human tRNAg& previously sequenced (Gupta et al., 1979). The glycine tRNA gene I has a striking 96% homology with a human tRNA& gene previously reported from our laboratory (Shortridge et al., 1985), but has only a 68 y0 homology with a human placental tRNAz& (Gupta et al., 1980). The sequence homologies of tRNAg$& gene I to other glycine tRNAs (Sprinzl et al., 1985b) or tRNA genes (Sprinzl et al., 1985a) with an anticodon of GCC are: E. coil tRNA (58x), yeast tRNA (66x), wheat germ tRNA (77%), Bombyx mori tRNA (95x), Bacillus subtilis tRNA gene (68%) Euglena grads chloroplast tRNA gene (63x), and Drosophila melanogaster tRNA gene (80%). Lawrence et al. (1985) made the observation that three difYerent types of repetitive sequence elements in higher eukaryotes may be retroposons that were ultimately derived from several tRNA molecules, and two of these elements have a great similarity to human tRNAg&. In particular, the tRNA$$ is quite homologous to the identifier sequence elements located in the intervening sequences of brain-specific genes in rats (Sutcliffe et al., 1984). Initiation of transcription by RNA polymerase III always occurs at a Pu residue, usually in the sequence Py-Pu-Py, 3 to 10 nt upstream from the structural gene (Ciliberto et al., 1983), whereas termination of transcription occurs in a stretch of at least four consecutive T nt in the 3’-flanking region of the gene (Bogenhagen and Brown, 1981). Since the te~ination of tr~sc~ption of both genes I and II would most likely occur in the five consecutive T nt in the 3’-flanking regions (from nt 356-360, and nt 1184-l 188 of Fig. 3), 17 and 16 nt, respectively, from the 3’-end of the tRNA structural genes, and since the putative precursor tRNAs can be estimated to be about 93-95 nt in length, the initiation of transcription should occur about 6-8 nt upstream from these structural genes. This would seem to indicate that initiation could occur at either the A residue or G residue at positions - 7 or - 6, respectively, in the identical sequence of eight nt (5’-CAGCTGGA-3’) immediately preceding the coding regions of both genes. This assumption is partially supported by the oligonucleotide the unique presence of

5’-GGAGC-3’ (shown schematically as spot 6 of Fig. Sb, and as spot 10 of Fig. 6d), derived from the 5’-terminal regions of both the putative tRNAg& precursor transcripts. This assumption is supported further by the absence of any other unique oligonucleotides in the RNase T, and RNase A fingerprints of the precursors which would be transcribed from farther upstream of the coding regions of both genes, The coding region of tRNAg& gene I contains a characteristic internal split promoter sequence (Ciliberto et al., 1983), with a block A sequence of 5’-TGGTTCAGTGG-3’ (residues 8-18 in Fig. 4) and a block B sequence of 5’-G’ITCGATTC-3’ (residues 51-59 in Fig. 4). In contrast, tRNA$& gene II has a T residue substituted (resulting from a C to T transition) at nt 54 of Fig. 4, resulting in the sequence TTTG within the block B internal promoter of gene II instead of the highly invariant TTCG sequence that corresponds to the T+CG sequence of mature tRNAs (Rich and RajBhandary, 1976). The second base substitution in tRNAg$, gene II at nt 67 of Fig. 4 does not occur at the position of a conserved nt, and would not be expected to have any unusual effects on the expression of gene II, especially since the G67 could form a bp with the U4 (corresponding to nt 4 in Fig. 4) of a gene II transcript. As can be seen from Fig. 5b, tRNAg$, gene I is accurately and efficiently transcribed by RNA polymerase III in an in vitro HeLa cell extract. During the incubation period, apparent precursor tRNAg$, transcripts undergo processing, such that a preponderance of the mature-sized tRNA product was observed toward the end of the incubation period. In GCC gene II was transcribed very contrast, tRNAGiy inefficiently, resulting in a six-fold reduction in the amount of putative precursor transcript derived from gene II, relative to that of gene I. In addition, essentially no mature-sized tRNA product derived from gene II was observed during the incubation period, as shown in Fig. 5b. Since there is a significant difference between genes I and II in that gene II has T54 at an otherwise invariant C residue in the block B internal promoter sequence, there is a great likelihood that this single nt transition could drastically decrease the efficiency of initiation of transcription of gene II relative to gene I, and also perhaps lead to the decreased stability of the mature tRNA product,

165

since essentially no mature tRNA product accumulated. Since a U residue would occur at nt 54 of a mature tRNAg& derived from gene II, this tRNA could potentially be inherently unstable due to the lack of the invariant G18. C54 tertiary bp which (Rich and occurs in virtually all tRNAs RajBhandary, 1976). Indeed, similar C to T mutations at this locus of two tRNA genes (Koski et al., 1980; Traboni et al., 1984) and also of an &-like element (Shimada et al., 1984) drastically diminish the level of transcription of these genes. This type of C to T transition also results in a lack of processing and in the degradation of a mutant precursor tRNA, due to its apparent inherent instability, most likely attributed to a lack of the crucial G. C tertiary bp (Traboni et al., 1984). Thus, as human tRNAg$, gene II contains a nt substitution that apparently diminishes drastically its gene expression, this tRNA gene would more properly be classified as a tRNA pseudogene. At least three putative Alu-like elements were detected on the 3’ side of tRNAg& gene II in the human DNA insert of AhGly3, with the nearest one being within 1.5 kb of gene II in Fig. 2a. HammarStrom et al. (1984) have proposed that Ah elements in the vicinity of a U2 RNA pseudogene may have relaxed or interfered with crucial cellular gene correction mechanisms, thereby allowing base substitutions to occur such that a previously normal U2 RNA gene was converted into the pseudogene. It is possible that a similar explanation could be applied to tRNA& gene II to account for its two base substitutions, and that the T54 substitution in the block B promoter of gene II, in particular, rendered it a pseudogene. The glycine tRNA genes I and II have identical sequences of eight nt (5’-CAGCTGGA-3’) immediately preceding the coding region of the two genes, and also have very similar stretches of about 10 nt (5’-CAACAAAACA-3’, residues 235-244 5’-CAACTAAAAACA-3’) residues and 1075-1086, shown in Fig. 3), centered at about - 32 and - 20 upstream from genes I and II. respectively. The 5’-flanking regions of several lower eukaryotic tRNA genes (Larson et al., 1983; Schaack et al., 1984; Johnson and Raymond, 1984) and a silkworm 5s RNA gene (Morton and Sprague, 1984) have been implicated as being regulatory elements for the proper initiation of transcription. Recently, the 5’-

flanking sequences about - 5 to - 40 nt upstream of mammalian opal suppressor tRNA genes have also been implicated as being transcriptional control elements (O’Neill et al., 1985; Pratt et al., 1985). It is tempting to speculate that these homologous regions of glycine tRNA,,, genes I and II have analogous functions in the control of expression of these genes. However, there are no similar sequences in the 5 ‘-flanking region of a human glycine tRNA,,, gene (Shortridge et al., 1985) or in other eukaryotic glycine tRNA genes (Sprinzl et al., 1985a). Thus, further studies will be required to ascertain if these short homologous sequences serve as regulatory elements in the transcription of tRNAg& gene I.

ACKNOWLEDGEMENTS

We are grateful to Drs. Jerry L. Slightom and Oliver Smithies of the University of Wisconsin for providing us with the human gene library. Also, we wish to acknowledge the support of Dr. Bernard S. Dudock of SUNY at Stony Brook, in whose laboratory some of the preliminary work was done. This work was supported by Grant GM30671 from the National Institutes of Health, Grant B-856 from The Robert A. Welch Foundation, NIH Grant BRSG2S07 RR07195, a grant from Research Corporation, and North Texas State University Organized Research Funds.

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