Complete synthesis and transcription in vitro of a gene coding for human ribosomal 5S RNA

Gene. 64 (1988) 77-85

77

Elsevier GEN 02332

Complete synthesis and transcription (Ribosomal

5s RNA;

III-transcription

in vitro of a gene coding for human rihosomal 5s RNA

gene expression;

in vitro transcription;

HeLa cell extract;

RNA polymerase

III;

Pol

factors)

Edgar Wingender”, Ronald Franka, Helmut Bliicker”, Lingru Wang b, Dieter Jahnb and Klaus H. Seifart’ ‘I Gesellschaft ftir Biotechnologische Forschung, D-3300 Braunschweig (F. R. G.) Tel. 0531/6181206. biologic und Tumorforschung, Philipps-Universitiit, D-3550 Mmburg (F.R. G.) Received

17 November

Accepted

4 December

1987

Received

by publisher

12 January

and Institut ftir Molekular-

1987 1988

SUMMARY

The gene coding for the major human ribosomal 5s RNA was chemically synthesized and cloned into a pUC13 vector. This approach was taken, because attempts to isolate the human 5S gene have thus far yielded either pseudogenes or variant 5s genes of unknown function. The synthetic human gene was transcribed by RNA polymerase III either in a crude HeLa cell extract or in a system reconstituted from partially purified transcription factors. Comparative studies with the Xenopus luevis somatic 5s gene show that the human gene is transcribed with similar fidelity and an efficiency of about 80% under optimal conditions. The time-course of transcription and optimal concentrations of template and transcription factors were found to be similar for both genes studied. The synthetic gene described may prove useful to study its interaction with human transcription

factors

in a homologous

system.

INTRODUCTION

Transcription of the gene for 5s rRNA by RNA polymerase III has proven to be an extremely useful

Correspondence to: Dr. K.H.

Seifart,

biologie und Tumorforschung,

Karl-von-Frisch-Strasse,

Marburg

(F.R.G.)

Abbreviations:

bp, base pair(s); liquid

deoxynucleotide; PMSF,

D-3550

DNA coding

h, human;

polyacrylamide fluoride;

for 5S RNA;

RNA.

I’)88 El.xGx

HEPES.

sulfonic acid; HPLC,

chromatography;

PAGE,

037X-l 119!88~$03.50 0

DTT, dithiothreitol;

N’-2-ethane

phenylmethylsulfonyl

5S DNA,

Rir Molekular-

Tel. 06421-285013.

hydroxyethylpiperazine performance

Institut

oligo,

higholigo-

gel electrophoresis;

TF, transcription 5S rRNA,

factor;

ribosomal

5S

model for the characterisation of the transcription factors involved and for the development of concepts regarding the mechanism of gene regulation in eukaryotic cells. Simultaneously it has become increasingly apparent that the development of homologous systems may be essential for the appropriate characterisation of promoter sequences and factors interacting with them. While TF IIIA, IIIB and IIIC have been isolated and partially purified from human cells (Segall et al., 1980; Wingender et al., 1986; Boulanger et al., 1987) the homologous gene for 5s rRNA has hitherto not been isolated in a transcriptionally active form from genomic DNA. A DNA sequence from a human library, which hybridized to 5s RNA and bound to

Science Publlshcrs B.V. (Bmmcdical Dwision)

78

TF IIIA was not transcribed change in the internal Roeder,

in vitro, due to a base

control

region (Emerson

1984) and it possibly

represents

of a large family of pseudogenes, of unknown

function

(Doran

and

a member

or variant

5S genes

et al., 1987).

In view ofthe outlined necessity to obtain a human 5s rRNA

gene of correct

ficulties associated

library, we synthesized for

the

major

Weissman, homologous

sequence

with its isolation a nucleotide

human

5s

1967) to investigate

and

factors

denaturing

temperatures

conditions

and ligated at

with T4 DNA ligase.

Finally the reaction

products

were checked

by PAGE

(see Fig. 2); all this according

procedures

detailed

elsewhere

(Frank

again to the

et al., 1983;

1987). (b) Cloning and sequencing

sequence coding

rRNA

(Forget

and

its transcription

by a

involved.

Expression of chemically synthesized eukaryotic genes in eukaryotic cells or cell extracts has been hitherto restricted to a few examples of synthetic structural genes due to the complicated and often unknown structure of their promoters. To express a synthetic human interferon-a, gene in murine cells, e.g., these problems were partially circumvented by using a heterologous viral promoter (Coulombe and Skup, 1986). Genes transcribed by RNA polymerase III, however, harbour their own promoters inside the transcribed region. Here we present results showing that a synthetic human 5S rRNA gene is correctly and productively transcribed in an in vitro system reconstituted from a HeLa cellular extract. This proves that for the expression of the human 5S RNA gene, 5’-flanking sequences are not essential, although they might exert additional regulating influences.

MATERIALS

under

the dif-

from a human

cell extract, and to study its interaction

with the transcription

PAGE elevated

AND METHODS

Using

the single-stranded

double

strand

BumHI

protruding

that

were

ends of the synthetic designed

as Sal1

ends, respectively,

gene was cloned into a pUCl3

and

the synthetic

vector. After ligation,

the plasmid was transfo~ed into ~,~~~e~ic~ja coli strain BMH 7 I-18, white clones were selected and checked by restriction analysis. Inserts from the plasmid DNA of positive clones were excised with Hind111 + EcoRI, recloned into phage M13mp18 and sequenced according to Sanger et al. (1977). (c) Preparation of DNA and cellular extracts Biochemicals were reagent grade from Boehringer Mannheim. The plasmids used in this study were isolated in their superhelical form as described (Currier and Nester, 1976) and purified by sucrose density gradient centrifugation. Plasmids pH5S and pX ls5S contained single copies of the synthetic human 5S gene and the X. laevis somatic 5S gene in pUC13 (2797 bp) and pUC I9 (2884 bp), respectively. The cytoplasmic HeLa cell extract (SIOO) was prepared from 20 liter suspension cultures with the titer of 4-5 x lo5 cells/ml, as described previously (Gruissem et al., 1981), and contained approx. 15 mg proteiniml.

(a) Gene design and synthesis Oligodeoxynucleotides comprising basically the coding sequence of the human 5S rRNA, its complementary strand, and ‘sticky ends’ for the restriction enzymes Sal I and BamHI were designed with the SYNGEN option of the GENMON computer programme package (see Fig. 1). synthesised with cellulose disks as segmental supports, purified after deprotection over reversed-phase (disposable cartridges) and ion-exchange (HPLC) materials, labelled at their 5 ’ -termini with [ y-‘*PI ATP and T4 polynucleotide kinase, analysed by sizing on a 20”,

(d) In vitro synthesis and electrophoretic RNA

analyses of

The in vitro transcription reaction mixtures were incubated at 30°C for 1 h unless stated otherwise and contained in a total volume of 50 11 0.025 mM and 10 /iCi [ z-~‘P] GTP (440 Ci/mmol, Amersham), 0.5 mM each of ATP, UTP and CTP 20 mM Tris ’ HCI, pH 7.9, 60 mM KCl, 5 mM MgClz, 3 mM DTT, IO?,; (v/v) glycerol, variable amounts of plasmid DNA and 15 ~1 SlOO. The in vitro transcription products were purified

19

and analyzed acrylamide

by PAGE, gels, and

using 7 M urea-lo”/:, subsequent

poly-

135 or 150 mM

autoradiography

through

overnight as described (Gruissem and Seifart, 1982). The region of the gel containing the RNA was excised

tions fractions

and quantitated

through,

by Cerenkov

radiation.

of transcription

All operations stated

were performed

otherwise.

transcription of HeLa

For

unless

of individual

trated by ammonium

culture

against

(about

B and

stored

at

in preparation).

two consecutive steps of buffer I containing 0.35 M KC1 (fraction B; approx. 1.3 mg protein~ml) and 0.6 M KC1 (fraction C; approx. 0.5 mg protein/ml). Fractions B and C contain TFIIIB and TFIIIC, respectively, and both fractions contain RNA polymerase III. The enzyme was removed from these fractions by dialysing against buffer II (20 mM Tris +HCI, pH 7.9,5 mM MgCl,, 3.0 mM DTT and 0.2 mM PMSF) containing 20% glycerol and either

RESULTS

AND DISCUSSION

(a) Gene synthesis The

chemical

and cloning synthesis

of oligos

as building

blocks for a relatively long DNA duplex comprising a complete gene is most efficiently carried out by simultaneous synthesis of adjacent fragments of

30 I

40 I

3

1

and

dialysed

60 mM KC1 and IO?/;, - 80°C. Human TFIIIA

(hTFIIIA), contained in fraction A (see above) was purified by additional chromatographic steps involving rechromato~aphy on phosphocellulose as will appropriately be published elsewhere (L. W., R. Waldschmidt, D. J., E. W. and K.H. S., manuscript

washed with the same buffer and the flow-through was collected (fraction A; approx. 3.0 mg protein/ml). The column was subsequently eluted with

20 I

sulfate precipitation,

buffer II containing

glycerol

pH 7.9, 3.0 mM DTT, 0.2 mM PMSF, 20% glycerol) containing 0.1 M KCl. The column was

I

III is retained

(Segall et al., 1980). Fractions

at 0-4°C

in suspension

10

RNA-polymerase

in the flow-

nation and from RNA polymerase III, but contained several other proteins. These fractions were concen-

15 mg protein/ml) was applied to a 30 ml; 2.6 x 10 cm phosphocellulose column (Whatman Pl I in its sodium form) in buffer I(20 mM HEPES,

I

bed

Under these condi-

B and C are contained

whereas

passage

C were assayed to be free of mutual cross contami-

the isolation

1

and

protein/ml

factors

factors the SIOO extract from 25 liters

cells grown

sulfate, (1 mg

volume) at these ionic strengths.

on the column (e) Fractionation

~monium

DEAE-Sephadex

50 I

60 I

5

7

TCGACGTCTACGGCCATACCACCCTGAACGCGCCCGATCTCGTCTGATCTC(S’WXT~T~ -TGCCGGTATGGTGGGACTTGCGCGGGCTAGAGCAGACTAGAGCCTTCGAl-KGT2

4

70

80

I

I

90

1

100

110

I

I

11

9 GCCTGGTTAGTACTTWTGGGAGA

..

6

120

I 15

13

CCGCCTGGGMTACCGGGTGCTGTAGGCTTTTAGACTlTTG

CGGACCAATCATGAACCTACCCTCTGGCGGACCCTTATGGCCCACGACATCCGAAAATCTGAAAACCTAG 0

10

Fig. I. Synthesis S. horedis position Sequence

of the human

termination

sequence

5S rRNA gene. Both strands and a BantHI

in the DNA duplex). The overlappings numbering

refers to the transcription

14

12 of the gene for human

site complement

were synthesised

of complementary start point

( = 1).

segments

5S rRNA

as adjacent

were optimised

flanked fragments

by a Sal1 site complement, (numbered

using a computer

according

programme

an

to their

(SYNGEN).

each strand which cover the complete sequence. For optimal hybridisation of complementary fragments

play only a single deviation

prior to their ligation, they have to be properly defined to achieve most stable and unique overlaps.

center

This was achieved the

computer

using an option

programme

package

which runs on VAX computers the operating (Frank For

(SYNGEN)

here, the nontranscribed

structed

of seven,

the transcribed

oligos (Fig. 1). These

fragments

53 which is located

and has recently

been shown

in the

(Pieler et al., 1987) to be important

for

GENMON

and IBM-PC

human

reported

in position

of the boxA homologue

con-

of

SS rRNA strand strand

/

under

systems VMS and DOS, respectively

et al., 1987). synthesis of the

trol region

within the internal

A

I

8

gene

was conof eight

were synthesized

using the filter method, purified and processed as described (Frank et al., 1983; 1987). Analysis of the individual oligos by gel electrophoresis revealed some minor contaminations in an overexposed autoradiograph (Fig. 2A). Nevertheless, they can be efficiently ligated to the correct product in a single reaction step (Fig. 2B) due to their proper design allowing ligation at elevated temperature and, thereby, suppressing any side-reactions. Referring to the nontranscribed strand, the 5’ terminus of the gene was designed to be compatible to a SulI restriction site. The subsequent region coding for the human 5s RNA was taken from the known RNA sequence (Forget and Weissman, 1967; Erdmann and Wolters, 1986). At the 3’ end of this region, the transcription termination sequence of a Xenopus borealis 5s rRNA gene (oocyte-type gene 1; Korn and Brown, 1981) was added which comprises two clusters of four thymidine residues each. These short T-stretches have been shown to represent efficient termination signals for RNA polymerase III transcription (Bogenhagen and Brown, 1981). The 3’ terminus consists of a sequence compatible to a BarnHI restriction site. Thus, the 5s rRNA gene complemented by a termination sequence was suitable to be cloned between the ScllI and the BumHI sites of the vector pUC13 (Messing, 1983). Subsequently, the synthetic gene sequence was verified using the sequencing method of Sanger et al. (1977). This sequence was compared to other 5s RNA genes previously employed for in vitro transcription studies, i.e., the oocyte-type 5s RNA gene from X. borealis (Korn and Brown, 1981) and theX. luevis somatic 5s gene (Brownlee et al., 1972; Fig. 3). The X. Iuevis somatic and the synthetic human gene dis-

m 234m 194mD

'

118c,

721)

1 Fig. 2. (A) Sizing of the synthetic

oligos (in the order

from left to right) on a 20”, polyacrylamide urea; lanes: A, the A ladder

starting

starting

of the one-step

with T,. (B) Analysis

to I5 on a IO”,, non-denaturing sample

of the crude ligation

4O”C, I h at 37”C, markers,

7M

with A,: T, the T ladder ligation of oligos I

polyacrylamide

mixture

I to 15

gel containing

gel; laws:

after incubation

1h at 3O’C, and overnight

(5’-“P-labelled

2

HueHI-fragments

for

I,

I h at

at 15’C; 2, M,

of 4x174).

81

1

20

10

hwn 5s RNA

GTCTACGGCCATACCACCCTGAACGCGCCCGATCTCGTCT l l ******** *********** l l *************

Xls

GCCTACGGCCACACCACCCTGAAAGTGCCCGATCTCGTCT

5s RNA

60

70

80

Xbo 5s RNA

GATCTCGGAAGCGATGCAGGGCCGGGCCTGGTI-AGTACCT l *********** * l ***** ********a****** l

hum 5s RNA

GATCTCGGAAGCTAAGCAGGGTCGGGCCTGGlTAGTACTT l *********** ***************************

Xls

GATCTCGGMGCCAAGCAGGGTCGGGCCTGGlTAGTAClT

5s RNA

90

110

100

120

Xbo 5s RNA

GGATGGGAGACCGCCTGGGAATACCAGGTGTCGTAGGCl-T l ************************ **** ********

hum 5s RNA

GGATGGGAGACCGCCTGGGAATACCGGGTGCTGTAGGClT ******** ********a**************** ****

Xls

GGATGGGAGACCGCCTGGGAATACC

5s RNA

comparison Sequences

AGGTGTCGTAGGCTT

of the gene for human 5s RNA (hum 5s RNA) with the genes for X. borealis oocyte-specific 1981) and for X. laevis somatic

5s RNA; Korn and Brown, by an asterisk.

4.0

GCCTACGGCCACACCCCCCTGAAAGTGCCCGATCTCGTCT l l ******** *** l ****** * **************

50

Fig. 3. Sequence

30

Xbo 5s RNA

of the non-coding

strands

5s RNA (Xls 5s RNA; Brownlee

are given; numbering

et al., 1972). Identical

refers to the transcription

5s RNA (Xbo

residues

are marked

start point ( = 1).

transcription efficiency (McConkey and Bogenhagen, 1987). The oocyte gene promoter differs in this and two additional positions from both these somatic genes.

5s-

(b) Time course of the in vitro transcription of the Xenopus Zuevis somatic and synthetic human SS-genes by a HeLa cell extract A human genomic clone, containing the 5s RNA sequence with one base change in the internal control region was not transcribed in vitro (Emerson

0

J

1

8 2

3

10

5

6

template. times

represent

products

after 5, 10,20,30,45

20

30 Time

45 1min. 1

60

containing counting.

reactions

were stopped

were separated

gels and visualised 5s RNA

insert, part A) or human

*

of the X. /aeviy somatic 5s RNA gene (o--(>). with nucle-

labelled GTP and 20 pg/ml of the indicated

The transcription

and RNA

polyacrylamide l-6

human

HeLa cell extract (SlOO) was incubated

oside triphosphates, 4

for transcription

or the synthetic

Cytoplasmic

-Y

1:

5

Fig. 4. Time course (M)

by autoradiography.

synthesised synthetic

Lanes

from the X. luevis (upper

5s gene (lower insert, part B)

and 60 min, respectively.

5s RNA were excised

at different

on 7 M urea-lo”,

Radioactive

and quantified

bands

by Cerenkov

82

and Roeder, analyse human the

1984). It was therefore

the transcription gene described

established

capacity

of transcription

5s gene. As was previously

(Pelham

and Brown,

scription

of the latter gene displays

to

gene is transcribed product

of the synthetic

here and to compare

kinetics

X. laevis somatic

of interest

it with

Xenopus somatic

of the

thesis

shown

total synthesis

1980; Jahn et al., 1987) trana clear time-lag,

with similar fidelity, yielding

identical

in size to that synthesised

5s gene. The rates of RNA

from both

somewhat

genes

are comparable,

has repeatedly

lower

(about

Figs.

syn-

although

been observed

20%,

a

on the

to be

4 and

5) in

to the Xenopus gene. The reason for this

comparison

presumably due to the delayed incorporation of TFIIIB into the transcription complex (Bieker et al.,

is not clear, but could be attributed to the lack of flanking sequences, possibly modulating the tran-

1985). As shown in Fig. 4, the synthetic

scription

gene

is productively

transcribed

human

in a HeLa

5s cell

extract and the formation of 5s RNA displays a very similar time course, including the time-lag mentioned above. (c) Influence of template concentration To investigate whether the human 5s gene is accepted by transcription factors in the HeLa cell extract with comparable affinity, a DNA titration was conducted in comparison to theX. laevis somatic 5s gene. As is demonstrated

in Fig. 5 the human

ever,

efficiency

that

observed

the

of this gene. It is evident,

DNA-optimum

for transcription

how-

characteristically

of the

5s

gene

and

attributed to the individual dispersion of TFIIIA and TFIIIC to separate genes at high DNA concentration without formation of productive preinitiation complexes (Wingender et al., 1984), is similar for both genes investigated. The absolute position of this optimum is different from that observed for the oocyte-type 5s gene from X. borealis (Gruissem et al., 1981; Jahn et al., 1987). This might be due to several additional base exchanges in the promoter region (50-90) of the latter gene when compared to

l

\ \ 0

\ \ \ \

1

\ \

\ \

\ \

\ \

\ \

\

’\

‘1

‘. \ \\ \

’\

-

a.,

.”

d”

DNA Fig. 5. Transcription GTP and increasing and quantitation

dependency amounts

on template

ofX. luevis (M

were as described

concentration.

&-hnll

HeLa S 100 was incubated

and insert A) or synthetic

in the legend to Fig. 2.

-

for 1 h with nucleotide

human 5s DNA (o-_O

triphosphates,

and insert B). Product

labclled analysis

83

either the X. laevis somatic

or the synthetic

human

gene (Fig. 3) causing less stable protein-DNA

inter-

actions

(Wormington

et al., 198 1).

(d) Influence of TFIIIA Binding of TFIIIA

and TFIIIC

from HeLa cells

to the internal

control region of

the 5s gene is the primary event for the formation transcription

complexes

on this gene. Although

of the

exact function of TFIIIC in this context is still not fully understood, recent investigations by Pieler et al. (1987) have demonstrated

interactions

of this factor

with boxA and probably also with boxC of the internal control region. It was therefore important to analyse the effect ofthese factors on the transcription

Fig. 6. Titration stituted

of the synthetic human 55 gene. It was found (Fig. 6) that 5S RNA synthesis from both genes increases with rising concentrations ofhTFIIIA up to a certain point and then decreases with very high concen-

genes in question. A comparable optimum is not observed for the titration of hTFIIIC (Fig. 7). It was observed that rising hTFIIIC concentrations increased 5s RNA formation without a subsequent decline. As is shown in Fig. 7, maximal rates of 5S RNA synthesis are

Fig. 7. Titration

of increasing

amounts

RNA gene (part B). Transcription ofTFIll.A,

10.0 ,ul ofTFIIIB,

The amounts

of hTFIIIf

of hTFIItC

factors

in a reconstituted

were

purilied

AND

METHODS,

TFIIIC,

varying amounts

template

were incubated

hTFIIIA

employed

ofTFIIIC

amounts

of hTFIIIA

respectively.

from

HeLa

in a recon-

(part A) OF synthetic ceils

factors as

IIIA, IIIB

described

section e. Ten yl ofTFIIIB, ofTFIIIA for

in

IO ~1 of

and 10 p’g DNA/ml

of either

1h in all assays. The amounts

of

were: in part A (lanes 1-5) O.OS,O.I, 0.5. 1.0,

5.0 ~1, respectively,

and in part B (lanes l-4) 0.05,0.5,

Quantitation

I .O. 5.0 ~1,

of 5s RNA was as in Fig. 2.

possibly reached at lower concentrations of/zTFIIIC in the case of the Xenopus gene. Footprint analyses (not shown; manuscript in prep~ation) proved, however, that hTFIIIC has little or no effect on the affinity of hTFIIIA binding to the human gene.

system with the X. iuevis somatic

were purified from HeLa celis as described

varying concentrations employed

IIIC

MATERIALS

trations of hTFIIIA. The basic phenomenon is similar for both genes, although the exact position of the optimum is slightly different, which could indicate an altered interaction of this factor with the two

of increasing

with the X. lnevis somatic

5s RNA gene (part B). Transcription

human and

system

and 5 ,ugjml ofeither

in

MATERIALS

DNA template

were 0.1, 0.5, 1, 2.5, 5, 7.5. 10, 20 and 50~1 in lanes

l-9,

(part A) or synthetic AND

METHODS,

were incubated

respectively.

human

5s

section e. One iti for 1 h in all assays.

84

(e) Conclusions

ACKNOWLEDGEMENTS

A gene coding for the major human chemically

synthesized.

5S rRNA was

This gene deviates

X. luevis somatic 5s gene, frequently studies, in eight positions.

from the

used for in vitro

One of them (at position

53) is located within the A box of the internal

control

region (Pieler et al., 1987). Although in a degenerate position of the BoxA consensus sequence, derived from either the tRNA genes (Ciliberto eukaryotic

5s RNA sequences

is apparently

important

We

acknowledge

Deutsche

financial

support

Forschungsgemeinschaft

conducted

at the University

of

the

to the research

of Marburg

and the

expert technical assistance of Frauke Seifart, Ursula Kopiniak and Verena Buckow (Marburg) as well as Helga Krause,

Christiane

Giesa,

Wiebke

Heikens

and Heiko Mielke (Braunschweig).

et al., 1983) or

(Pieler et al., 1987), it

for transcription

efficiency

(McConkey and Bogenhagen, 1987). However, we did not observe enhanced transcription as reported by these authors for a CG-TA transition in that position of the X. borealis somatic 5s gene. Our results clearly show that the synthetic human 5S rRNA gene is correctly and efficiently transcribed by RNA polymerase III either in a crude HeLa cell extract or in a reconstituted system of partially purified transcription factors. This proves that flanking regions are not required for transcription of the human 5S gene as was previously shown for the X. borealis somatic gene (Bogenhagen et al., 1980; Sakonju et al., 1980). Nevertheless, these flanking sequences may exert either positively or negatively modulating influences, as has recently been shown for some point mutations in the upstream region of that gene by McConkey and Bogenhagen (1987). Differences in the flanking sequences between the genes compared in this study may account for the slightly lower transcription efficiency of the synthetic human gene (approx. 80% under optimal conditions) and may possibly overcome the otherwise positive C + T transition at position 53. The synthetic gene described may prove useful to study its interaction with human transcription factors in a homologous system. We are presently analysing whether the small differences observed in the factor concentrations required for optimal transcription of the two genes could reflect altered protein-DNA interactions. Preliminary results from comparative footprint experiments show an altered interaction of human TFIIIA with the human and X. laevis somatic 5S genes, respectively, re-emphasising the importance of homologous in vitro systems.

NOTE

After this manuscript had been submitted, our attention was attracted to the paper by Arnold et al. (1987) describing the isolation of a variant and a pseudogene for human 5S RNA from a genomic DNA library. These genes are transcribed with very low efficiency and these data re-emphasise the importance of obtaining an authentic human 5S RNA gene.

REFERENCES Arnold,

G.J., Kahnt,

B., Herrenknecht,

variant gene and a pseudogene

K. and Gross.

ally active in vitro. Gene 60 (1987) Bieker, T.L., Martin, rate-limiting 40 (1985)

137-144.

P.L. and Roeder,

intermediate

H.J.: A

for 5s RNA are transcriptionFormation

of a

in 5S RNA gene transcription.

R.G.:

Cell

119-127.

Bogenhagen,

D.F. and Brown,

Xenopus 5s DNA required

D.D.: Nucleotide

sequences

for transcription

termination.

S. and Brown,

D.D.:

m Cell

24 (1981) 261-270. Bogenhagen, region

D.F.,

Sakonju,

in the center

initiation

A control

of the 5s RNA gene directs

oftranscription,

specific

II. The 3’ border ofthe region. Ccl1

19 (1980) 27-35. Boulanger,

P.A., Yoshinaga,

properties

and

SK. and Berk. A.T.: DNA-binding

characterization

of human

transcription

factor III C2. J. Biol. Chem. 262 (1987) 1509X-I5 Brownlee,

G.G., Cartwright,

R.: The

nucleotide

E., McShane,

sequence

105.

T. and Williamson,

of somatic

5s

RNA

from

Xenopus laevis. FEBS Lett. 25 (1972) 8-12. Ciliberto,

G., Raugei,

R.: Common

G., Costanzo,

F., Dente,

and interchangeable

of genes transcribed

by RNA

elements

L. and Cortese, in the promoters

polymerase.

Cell 32 (1983)

725-733. Coulombe,

B. and Skup, D.: Expression

interferon-a, mammalian

gene

with

modified

of a synthetic nucleotide

cells. Gene 46 (1986) 89-95.

human

sequence

in

85

Currier,

T.C. and Nester,

circular Biocbem. Doran,

E.W.: Isolation

DNA ofhigh molecular

of covalently

closed

weight from bacteria.

Analyt.

sequences

Bingle,

W.H.

of two human

and

Roy,

K.L.:

The

5S rRNA pseudogenes.

nucleotide Nucl. Acids

Res. 15 ( 1987) 6297. Emerson,

B.M.

arrangement

and

Roeder,

R.C.:

Isolation

of active and inactive

V.A. and Waiters,

and 4.5s (suppl. Forget,

ribosomal

and

genomic

forms of mammalian

SS

B.C. and Weissman, R., Heikens,

synthesis

Nucl.

Res. 14

Acids

S.M.: Nucleotide

Simultaneous fragments: Enzymol.

W., Heisterberg-Moutsis, approach

of KB

G. and Blocker,

of oligonucleotides:

chemical segmental

A., Schwellnus,

synthesis

K. and

and biological

an efficient and complete

Blocker,

applications

H.:

of DNA

methodology.

Methods

M. and Seifart, K.H.: Transcription 5s RNA in a system

in vitro from HeLa cells. Eur. J. Biochem.

of

recon-

117 (1981)

is feedback-inhibited

D., Wingender, for various

formation

for cloning.

Methods

Enzymol.

H.R.B. and Brown, D.D.: A specific transcription

factor

Sci. USA 77 (1980) 4170-4174.

Pieler, T., Hamm,

J. and Roeder.

control

region

ments,

organized

spacing.

is composed

R.G.: The 5S gene internal

of three distinct

as two functional

sequence

domains

ele-

with variable

Cell 48 (1987) 91-100. S., Bogenhagen,

region

in the center

initiation

D.F. and

Brown,

D.D.:

A control

of the 5S RNA gene directs

of transcription,

I. The 5’ border ofthe

specific

region. Ceil

19 {1980) 13-25. F., Nicklen, S. and Coulson,

chain-terminating

inhibitors.

A.R.: DNA sequencing

Proc. Natl. Acad.

with

Sci. USA 74

(1977) 5463-5467. Segall, J., Matsui, RNA polymerase Wingender, lation

T. and Roeder,

for the accurate

R.G.: Multiple

transcription

factors

of purified

are

genes by

III. J. Biol. Chem. 255 (1980) 11986-I 1991.

E., Shi, X.P., Houpert, of a transcription

A. and Seifart,

complex

by HeLa

of 5S RNA genes 5S RNA.

J. Biol.

for ribosomal

K.H.:

Iso-

5S RNA.

K.H.: Transcription

class III genes towards

E., Jahn, D. and Seifart, K.H.: Association

polymerase

III with transcription

L.J. and Brown,

differ in parameters

comof

salt. J. Mol. Biol. 193 (1987)

D.D.: Nucleotide

borealis oocyte

5S DNA: comparison

several

eucaryotic

related

factors

of RNA

in the absence

of

DNA. J. Biol. Chem. 261 (1986) 1409-1413.

E. and Seifart,

and stability

Wingender,

Wormington,

W.M., Bogenhagen,

D.D.: A quantitative scription

sequence of sequences

of Xenopus that flank

genes. Cell 15 (198 1) 1145-l 156.

Communicated

D.F., Jordan,

E. and Brown,

assay for Xert~~pus 5s RNA gene tran-

in vitro. Cell 24 (1981) 809-817.

303-313. Kern,

binding.

EMBO J. 3 (1984) 1761-1768. W. and Seifart, K.H.: Transcription

Chem. 257 (1982) 1468-1472. plexes

and TFHIA

that can bind either the 5S RNA gene or 5S RNA. Proc. Natl.

required

154 (1978) 221-249.

W., Kotzerke,

in vitro Jahn,

on transcription

J.: New Ml3 vectors

Pelham,

Sanger,

Nucl. Acids Res. 11 (1983) 4365-4377.

407-415. Gruissem,

Messing,

Sakonju,

for the simultaneous

the cloned genes for ribosomal stituted

sequence

158 (1967) 1695-1699.

R., Meyerhans,

Gruissem,

5S, 5.8s

sequences.

of large numbers

solid supports,

effects

mutations

5S RNA gene can have

Mol. Cell. Biot. 7 (1987) 486-494.

Acad.

ofpublished

1986) rl-r59.

H.: A new general

Frank,

J.: Collection

RNA

cell 5S RNA. Science Frank.

D.F.: Transition

101 (1983) 20-78.

RNA genes. J. Biol. Chem. 259 (1984) 7916-7925. Erdmann,

G.A. and Bogenhagen,

within the Xenopus borealis somatic independent

76 (1976) 431-441.

J.L.,

McConkey,

by H.G. Zachau.