Two human tyrosine tRNA genes contain introns

Gene.42

101

(1986) 101-106

Elsevier GENE

1544

Two human tyrosine tRNA genes contain introns (Recombinant

DNA;

nucleotide

sequencing;

plasmids;

5’-flanking

region; Xenopus laevis DNA probe;

Ml3

and A-phage vectors)

James M. MacPherson

and Kenneth L. Roy*

* Depurtrnent of Microbiology, University of Alberta, Edmonton, Alberta (Canada T6G 2E9) Tel. (403)432-5610 (Received

September

(Revision

received

26th. 1985)

and accepted

December

12th, 1985)

SUMMARY

A human A-phage recombinant which contains at least four tRNA genes, has been isolated. Two DNA fragments were subcloned to give the recombinant plasmids pM6 and pM6128. Nucleotide sequence analysis showed that each plasmid contained a different tyrosine acceptor tRNA (tRNATy’) gene. Both tRNAT”’ genes are interrupted by 21-bp introns. These recombinant plasmids have been shown to direct the in vitro synthesis of tRNA-sized products in a HeLa cell transcription system.

INTRODUCTION

Information on the organization and structure of human tRNA genes is very limited. Of the nuclear tRNA genes in man only a few examples coding for asparagine (Ma et al., 1984), glutamine, lysine, leucine (Roy, et al., 1982), glutamic acid (Goddard et al., 1983) and methionine (Santos and Zasloff, 1981) have been described. Transfer RNA genes in higher eukaryotic organisms can occur clustered at one locus or as orphans scattered throughout the genome, not apparently * To whom

correspondence

and

reprint

requests

should

be

addressed. Abbreviations: nucleotide(s); polymerase tRNA;

bp, base pair(s); PolIk,

Klenow

kb, kilobases

(large)

I; ss, single stranded;

fragment

tRNATy’,

or 1000 bp; nt, of

E. coli

tyrosine

DNA

acceptor

u. unit(s).

0378-I I lY/X6,'$03.5(1 10 1986 Elsevw

Science Publishers

B V. (Biomedical

associated with other tRNA genes. Clustering of tRNA genes has been observed by Roy et al. (1982), who found three different human tRNA genes within a 1.3-kb length of DNA. In Xenopus laevis eight tRNA genes, including one for tyrosine, are located on a 3.18-kb DNA fragment which is tandemly repeated at least 100 times on a single chromosome (Fostel et al., 1984). Methionine tRNA genes, however, have been found at scattered locations in the human genome (Santos and Zasloff, 1981). The functional basis for these varied organizational patterns is not known. Introns have been found in tRNATy’ genes from yeast (Goodman et al., 1977) and X. laevis (Muller and Clarkson, 1980). Introns usually occur one bp 3’ to the anticodon, although variations are known (Del Rey et al., 1982; Ogden et al., 1984). Johnson and Abelson (1983) have demonstrated that a yeast tRNA”y’ gene intron is essential for the correct Division)

102

modification no major genes.

of the tRNA. function

Other than this example,

is known

In an investigation

into

for introns

in tRNA

the organization

and

structure of human tRNA genes, a human A-phage recombinant has been isolated which appears to contain

at least

four tRNA genes.

which genes are present been subcloned

and used for sequence

communication

describes

tRNATY’ genes,

both of which

21-bp

introns.

examples tRNA

To determine

small DNA fragments

We believe

of introns

the

sequences

have This

of two

are interrupted

these

in human

analysis.

by 23

to be the first

(and

198

mammalian)

genes. 2x

I 51

to pM6128

;40 127

to

pM6

120 10 09

EXPERIMENTAL

(a) Isolation

of IZHtM6 and construction

of sub-

065

clones

A human %-Charon 4A phage recombinant library, a gift of T. Maniatis (Lawn et al., 1978), was screened for tyrosine tRNA genes by the method of Benton and Davis (1977) as previously described (Buckland et al., 1983). The probe used was a 262bp HhaI DNA fragment of the 3.1%kb X. luevtv tRNA gene cluster, which had been subcloned, using Hind111 linkers, into pAT153 (Twigg and Sherratt, 1980; W. Lam and K.L.R., unpublished). This subcloned X. laevis tRNATY’ gene was excised and 3’ end “P-labeled (with PolIk) and used in all probing procedures. A human i-phage recombinant, 3.HtM6, was isolated. Fig. la shows the DNA fragments generated by a Hind111 digestion of 1.HtM6 which have been separated on a 1 y0 agarose gel. The 1.98-kb, 1..51-kb, 1.40-kb and 1.27-kb DNA fragments all hybridize strongly to the X. laevis tRNA.‘.Y’gene probe. Fig. lb illustrates the hybridization of the X. laevis tRNATy’ gene probe to the same Hind111 digest of 1HtM6. From the pattern obtained it would appear that /IHtM6 contains at least four tRNAgenes. The 1.51-kb and 1.40-kb DNA fragments have each been subcloned, using standard techniques, into the Hind111 site of pAT153 (Twigg and Sherratt, 1980) to give the recombinant plasmids pM6 128 and pM6, respectively.

035

kb

Fig. I. Restriction

enzyme digest ofi.HtM6

and hybridization

to

the .Y. /revi.c tRNA-rY’ gene probe. (A) A I.5 pg sampleoflHtM6 was diyested

with 4 u of HirldIII

2.5 h. The products stained with ethidium illumination. 3’-end

(B)The

labeled

been hybridized

in a total volume of 20 ~1 for

were separated

on a I”,, agarose

bromide for visualization Southern

(with Pollk) overnight

transfer

gel and

under ultraviolet

of (A) to which

X. /revis tRNA’Y’

gene probe

the has

at 67. C.

(b) Sequences of tRNATY’ and tRNAp’

Nucleotide sequence analysis of short hybridizing DNA fragments from pM6128 and pM6 revealed that they each contain a tRNA’lr’ gene as indicated by the anticodon sequence GTA (Fig. 2). Both tRNA,!?’ and tRNA,‘,’ gene coding regions have almost lOOo/, homology to the X. laevis tRNA7y’ gene (Mtiller and Clarkson, 1980) with one A to G transition at position 37. The tRNA:r’ gene also differed from both the tRNA:s’ gene and the X. luevi.7tRNATY’gene by a second G to A transition at position 78. This results in the loss of HinfI and

103

-70

-6.0

-50

-4.0

-3.0

PM6

GGATCTCCGGTGGTCCAGGGACTTGGCTTCCTC~ATTTGCAG~AGTCCAGTG

pM6128

TTGACTCCAGCGTTCCAAGGACTTGGCTTCCTC~ATTTGCGG~AGTCCAGTG TY-b -1. !

PM6

-20 -1.0 ‘9 20 3P ACCCAGCCTTAACAGTGTGCA~CTTCGATAGCTCAGCTGG~AGAGCGGAGGA

pM6128

.~TCCAGCTCTTGCAGCGTGCACE=CTTCGATAGCTCAGCTGGTAGAGCGGAGGA TYR,

TYR,

pM6 pM6128

CTGTA~TTGTACAGA~ATTTGCGGA,~TCCTTAGGTCGCTGGTTCGATTCCG

PM6

GCTCGAAGG&GCGCiTGACTCTTTiGCGCACAAT&C-3'

pM6128

GCTCGAAGGAjAGTGCCCGATGCTTTTGCATGCAATGC-3'

90

Fig. 2. The sequence

of the noncoding

strand

of the tRNA gene-containing

the tRNA genes is indicated

by the arrow.

5’.flanking

have been repeated

sequences

corresponds

which

to the 5’-terminal

110

100

The boxes indicate

120

regions

of plasmids

pM6 and pM6128.

the two exons of each gene. The horizontal

within the intron

of each gene (see EXPERIMENTAL,

nt ofthe mature tRNA has been designated

TYR,

number

The orientation

bars show the location

of of

section c). The nt which

1. All nt 3’ to this reference

point were given positive

\ alues

TaqI sites in the 3' end of this gene. The 3’ CCA terminus is not encoded in either gene. Beyond both tRNATy’ genes is a short T tract located 13 nt past the 3’ end of the genes. In both cases these are suggested transcription termination signals for RNA polymerase III (Bogenhagen and Brown, 1981). There is also considerable homology in the regions between the 3’ termini of the genes and the termination signals. (c) Introns

and the 5’-flanking

regions

The most striking feature of both tRNATy’ genes is the presence of a 21-bp intron located one base to the 3’side of the anticodon (Fig. 3). Both introns begin with the same nucleoside, A, and end with the same four-base sequence, GGAC. The homology between the two introns is not complete with only 14-bp being conserved. Fig. 4 shows the two tRNAs drawn in the familar cloverleaf structure. In each

case the intron contains a short sequence homologous to the anticodon and maintains the indicated splice sites in ss loops. Considerable homology

is present

between

the

5’-flanking sequences of tRNAF’ and tRNA7’ genes. Within the first 60 bp immediately preceding the genes there are four regions, -57 to -36, -34 to -22, -20 to -16 +rd -6 to -2, with complete homology. A short lo-bp sequence located within the tRNA7’ intron at + 48 to + 57 is identical to position -42 to -33 of this gene’s 5’-flanking sequence. A similar sequence occurs in the tRNAF’ gene; however, there is not as extensive a duplication of sequences,

as only 7 of the 10 nt are identical.

(d) In vitro transcription

of pM6 and pM6128

In vitro transcription of both tRNATy’ genes in a HeLa cell extract showed the expression of tRNAsized products and presumed precursors (not

G

GATC

A

T

C

CG3 C TTA CG GTE CTG TCG AG CTT ATC

0’

_a c

z

z

0

a l-

z-

TAG CT GI AC

-

GG 5’

AC

A

tRNA;Y’ Fig. 3. Autoradiograph Overlapping

of 64, polyacrylamide,

HaeIII

of either Ml3mplO nation procedure

and Sau3A or Ml3mpl

(Sanger

DNA fragment

8.3 M urea sequencing

DNA fragments

I (Messing,

1983). These were sequenced,

et al.. 1977). The gels shown illustrate

from pM6128.

The introns

and anticodons

A

GG

u

cucGAU

(AC) are indicated

by brackets.

uc

shown). The extent of RNA synthesis directed by pM6128 was about sixfold higher than that synthesized from pM6.

*cuCGA

DISCUSSION

E:

G

C

A U / AGG”G C ‘AC ,,” IJ GCAG ACA VA p G

“““G GGAAAC

‘UG

‘W

,.NEq’

.aNA;“~

sequences

of the tRNAs

pM6128. The CCA terminus added for completeness. indicated sequence.

splice could

encoded

(not encoded

The anticodon

also

is indicated

The small arrows

be spliced

by pM6

and

by the DNA) has been

sites to yield the mature

by the large arrows.

the tRNAs

from pM6 and a HrreIII

GGU~GAGcG

GC

The expected

by the dideoxy chain-termi-

DNA fragment

G

CA,u!c”,,”

Fig. 4. The

as templates,

GC

CGGCdJ”A*

:‘c

u

into the SmaI or BanzHI sites. respectively,

CG

GC UGGUUC CU G UAGG A”

AGAGCG

using both strands

strand ofa Sau3A

CG “A UA

:t

GC AU

the intron regions of tRNA{Y’ and tRNAzY’ genes.

the noncoding

A

CG “A

“CGA

gels spanning

from both pM6 and pM6128 were inserted

by a bracket. sequences indicate

to yield the same

are where

mature

Within the vertebrates a limited but increasing number of examples of tRNA genes has been described (Looney and Harding, 1983; Makowski et al., 1983; Rosen et al., 1984; Roy et al., 1982; Ma et al., 1984; Shibuya et al., 1982). Little is known about tRNA gene organization in higher eukaryotes. To expand our knowledge of tRNA gene organization and structure we have, therefore, with the use of a cloned X. luevis tRNAry’ gene, isolated two human tRNATy’ genes. Both tRNAF’ and tRNA7’ genes are 94-bp in

105

length.

There

is extensive

X. fuevis and human the tRNA7’

homology

tRNATy’

gene coding

between

the

genes. In particular,

sequence

has

a single

bp alteration from that of the X. laevis tRNATy’ gene. The most characteristic feature of these genes is the presence

of a 21-bp

intervening

located one bp 3’ to the anticodon

sequence

in contrast

to the

X. laevis tRNATy’ gene intron which begins adjacent to the anticodon. conservation

Although

of both

tRNATy’

gene exons

homology

between

the

REFERENCES

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sequences.

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