Vol.

166,

No.

January

BIOCHEMICAL

1, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

15,199O

Cloning

of a hsp70-related Midori

Laboratory

of Cell

gene expressed

Matsumoto'

11 Minamiooya,

in mouse spermatids

and Hirokazu

Mitsubishi

Biology,

Fujimoto'

Kasei

Machida,

43-49

Institute

Tokyo 194,

of Life

Sciences

Japan

Received November 18, 1989 A cDNA library of spermatids was screened by a differential hybridization in order to isolate genes expressed in haploid cells of A clone was found that encoded a protein the mouse male germ line. related to the heat shock protein 70. A genomic DNA clone comparable to this cDNA clone was also isolated from a mouse genomic library. This gene had only one continuous open reading frame capable of coding a 630 amino-acid protein. There was an excellent match of the sequence with the heat shock protein 70 family but a difference from any previous 70 kilodalton heat shock protein. A 2.7kb transcript derived from this gene was expressed in spermatids but not in other testicular germ cells and somatic tissues. We have referred to this gene as hsc70t. 0 1990 Academic Press, Inc. Spermatogenesis which

mitotic

is a complex

proliferation

and differentiation

spermatozoa.

The distinct of

the

spermiogenesis not

found

fundamental genetic

analysis

spermatozoa, identified

morphological

and supported cell

reprogramming

the

of

the

is followed

of haploid

spermatozoa

in any other

of developmental

of spermatogonia

spermatocytes

function

series

are

type

genes

involved

in

the

and mechanisms

of their

expression

to mature specialized

during

final

steps

of

of

proteins

that

are

Spermiogenesis

of

of

and

of gene expression. construction

by meiosis

feature

number

(1).

in

spermatids

obtained

by a large

events

may require

A prerequisite the

complex

spermiogenesis

'Present address: Department of Life Science, Tokyo Institute of Technology, Meguro, Tokyo ‘To whom correspondence and reprint requests Abbreviations: HSP70, heat shock protein 70 shock protein cognate gene.

should

to the

structure should

in be

be ensured.

Faculty of Sciences, 151, Japan. should be addressed. kilcdalton; hsc, heat 0006-291x/90

43

a

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproducrion in any form reserved.

Vol.

166, No. 1, 1990

Recombinant

DNA techniques

genes expressed have isolated expressed also

during

specifically and its

was examined

Materials

mammalian

in

AND BIOPHYSICAL

relationship level

molecular

spermatogenesis

(2,

homologous

spermatids.

at the

RESEARCH COMMUNICATIONS

have been used for

a mouse cDNA clone

isolated

family

BIOCHEMICAL

to

to other

3, 4). genes

hsp70

A genomic

cloning

DNA clone

Here,

we

which

is

has been

members of the hsp70

of DNA sequence

of

gene

analysis.

andMethods

Preparation of testicular germ cells: Sexually mature male mice were used for preparation of testicular germ cells by the two step method with collagenase and trypsin, as described previously (1). Centrifugal elutriation was performed on the testicular germ cells using a Beckman JE-6 elutriator roter (5). Cell culture and heat shock conditions: Mouse Ltk- cells were grown inEagle mini= essential medium with 10% fetal bovine serum at 37'C with 5% CO . For the heat shock treatment, the cells were kept at 44'C for 9 8 min and allowed to recover for 2.5hr at 37°C. Extraction of RNA: RNA was isolated from cells and tissues by the guanidium thioGnate/ CsCl method (6). Poly(A)+ RNA was selected through two cycles of oligo(dT) cellulose chromatography (6). Construction _cDNA library and a mouse genomic library: --of a spermatid Poly (A)+ RNA of early spermatids isolated by elutriation was used for construction of a cDNA library with lambda phage igist as a vector (6). The mouse genomic library (a gift from Dr. A. Matsushiro) was constructed in a lambda phage EMBL4 vector by partial digestion of 129/SvJ mouse DNA with EcoRI. DNA sequence analysis: DNA fragments were subcloned into a pUCl9 and Gleotide sequences were determined by Sanger's method (7). Northern and B slot-blot analysis: For Northern analyses poly(A)+ RNA was subjected to electrophoresis in 1% agarose-formaldehyde gel (6) and transferred to Biodine A nylone membrane (Nihon Pall, Japan). For the RNA slot blot analysis total RNA was transferred to nylon membrane using a microsample filtration manifold device (Schleicher and Schuell) ilters were hybridized with appropriate DNA probes labelled witi " P by Amersham multiprime DNA labelling system. Results

The

and Discussion

spermatid

hybridization prepared testes,

method from

which

spermatid isolated

cDNA library

probe

either

using

had no spermatids.

from spermatocytes

gave clear

indications

screened

32P-labelled

spermatids

were picked

was

or

immature

Several

and spermatids. expressed

(see below). 44

a differential

cDNA probes

and analyzed

of being

by

poly(A)+RNA

(17-day)

clones with

to

decapsulated

hybridized Northern

One of these abundantly

in

with

blots clones, haploid

of

the RNA HS2, cells

Vol.

166, No. 1, 1990

BlOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1 ATGG~~CCPAATAAAGGAATCGCGA~GGCA~GACCPGGGCACCAC~ACTCGTGCCTGGGCGTGTTCCAACAC~~~~AG~G~AG~TC HAANKGMAIGIDLCTTYSCVGVF~RG 9, A~~CGA~ACCAGGTCAA~~~AC~CCCCTGACTACG~~~~ACCGACACCAGCCGC~CATCGAGC~~~CAAGAACCAG~G RLIERSKNQ PDYVAFTDTS IADYQVNRTT 1.9,

GCCAn;AA~CCCAGAACACTGT~T~A~C~AACGTCTAAT~GCAGGAAGTTTAA~A~~GT~TGCAGTCAAAT~~~AA~~ LIGRKFNDPVVQSN A,,NPQNTVFDAKR

271 ;TG~C~TTTCAATGATCAATC~AAGCCGGCAAACCCAAGGTGAR;GT SMINEAGKPKVM

VSYKGEKKAFY

CAGAGGTTTTTTGGGCACCAATGTCACAACGTGTG;TC.$C

d $%pAgC~AT:W

361

~R$~W~:E484~~~;14~~~~~~~~~~ SSHVLTKMKETAEVFWAPHSQRV

451

AA~A~TCAGCGGCAAGCCACCAAGGATGCAGGTGTCA~GCAGGACTCAA~TG~GAGA~TAA~AA~AGCCCACGGCGCI‘CGCA INEPTAVA NDSQRQATKDAGVIAGLNVLR

5,l

n;GG~AGGTn;GATAAAGGAAGTCACG~GAGCGGCAC~~CA~T~GAC~GGG~GCAC~~GA~~TCCA~~G~CG VLIFDLGGGTFDLSI WARLDKGSHAERH

631

:TCW\CGACG~TCTrrAGTGAAGCULCGCGGCGACACG~C~GG~GGGGAGGACT~~CAACCGG~~AGCCACT~~GAGA DDASSVKPRGDTHLGGEDFDNRLVSHFVR

721 GI’CAAGAGGAAG~CAAGAAGGACAA’FCAAGCCAAGAACAAGCGCGCG~CGGCGG~CG~CGGC~G~AGAGGGCCAAGAG~CG VKRKHKKDNQAKNKRAVRRLRTACERAKRT 811

~GTCGTCCAGCACCCI\GGAAAC~~GA~GA~~~ATA~AGGG~~GACT~ACAC~CCA~A~~AGCACG~~AA DFYTSITRARFE LSSSTQANLEIDSLYEGI

901

GAGCK;n;TGCAGAC~ATTTAGAGG~CA~~AGCCCCC ELCADLFRGTLEPVE

991

ATlGTTCTAGTAGGGGGCTCCACCCGCAlCCCAAAGGTGCAMAAAlGCT~AGGACTA‘=T~AA IVLVGGSTRIPKVQKMLQDYFN

KSLRDAKMDKAKIHD -2

GACGGGATCTCAACAAGAGTA’l’2 RDLNKSI

1081

AATCCCGAn;AGG~GTCGC~ACGGI\GCFGChCTC~GGGAG~AT~M~GGCGACAM~AM~TACAG~TT~~~ LMGDKSEKVQDLLL NPDEAVAYGAAVQGAI

1171

TTG~CGTAGGCTCCC~GTCTCPAGGAT’FGGAGACAGC LDVAPLSLGLETAGGVMTVLI

1261

CAGACGAAGATCTrrACCAC~A~C~CAAC~GCCCGGG~A~A~Cffi~TACGAGGCAAACCATACCC~CAACAAC~~G QTKIFTTYSDNQPGVLIQCTRQTIPDNNLV

1351

GGGCC~~ACTTGACn;GAATAC~C~G~C~AGCQ GPFDLTGIPPAPSVPQIEVTFDIARNGILN

1441

GTTACGGCCATCCACAAGAGCACCGGCAAGGCCAACAAGA~AC~~AC~ACGACA~G~CGC~AGCA~GAG~GAT~A~CG VTAMDKSTGKANKITITNDKGRLSKEEIDS

1531

ATCGAGAGCCGGA~G~TACAAAC~GAGGA~AG~CAGAGGGAGAAAA~G~C~AAAA~C~TffiAA~~ACGC~TTAAT IESRTAYKREDEGQREKIAAKNALESYAFN

1621

A~AAGAGCGCPn;TGGTCATGAGGGTCTGAAGGACAAGA~ACGGAGTCCGATAAAA~AAAATA~GATAAA~CAA~AG~~~ NKSACGDEGLKDKITESDKKKILDKCNEVP

1711

T~~G~GAGTCAAACCCAGePGG[;TGAGAAAGTAGAGT~A~ATAAA~AAA~AAeM;GAAAATA~TGTAA~CG~~~~~CA FLLSQTQLA EKVEPDHKRKELENMCN

1601

AAA~TACCAGAGCGGATG~CCGGG~CCAC~TACGCCAGG~ATA~CCGGCAGGG~CCACAGGCC~ACC~TC~AG~A~T~DAT KLYQSGCTG LRQGSTGPT flbV R Q G I TAGCCFT~CAGAATn;CG~G~N~G~C~CTAGGCGAATT~A~A~T~~AACA~AATA~AT~T~AA~A~GGA

1891

KRNSTIPTK

GATCCCGATGG GAAGCTCGAACGCAC~TTTCACACCCCCACCCGYCGTCAGTCTATGA~CIC 1891P ACCA9 CCTAC~CACCAGTCCT’Jlj GA‘X?r TAcxAA~ACCA G CCCTCTTTAGA~C~~TGA~A~T~~TCT~A~ATTA~CACACCCCAC~ 2161 C~GTGTGTTGGTTATTTGTCTCTC~~TAAATT~;TTCCAAAGGAAACAA~C~CACTT*AT~AGCAGGC~TA~GAT~~~ f WlYA

Fis.

1. Nucleotide sequence of the mouse hsc70t gene and the deduced amino acid sequence, as determined from the genomic GE6 and the HS2 cDNA clones. A nucleotide sequence shown in a small letter was derived from the 5' end of the HS2 cDNA clone. Polyadenylation signal is underlined. PolyA; addition site of a polyadenylation tract, Bn; Ban1 site, BI; BemHI site.

analyses

DNA sequencing

database insert

of the with

terminal

an amino

untranslated

that

of the

a genomic region

and isolated they

acid

shared

that

searching

the region

Xenopus

HSP70

signal

(Fig.

DNA library

(BarnHI-EcoRI 2 positive

conunon restriction

homologous

to

a 1.9

kb

the

carboxyl

and a 3' untranslated

region

(4.5

x IO' of

the

clones)

using

HS2 cDNA clone

The restriction sites.

45

contained

sequence

1).

200bp)

clones.

the protein

HS2 clone

coding

a polyadenylation

We screened

probe

NBRF revealed

residues

including

and computer

mapping

The 3' untranslated

the

3'

as a showed probe

Vol.

166, No. 1, 1990

BIOCHEMICAL

of the HS2 cDNA detected fragment

length

sequencing provided for

of

(Fig. artifact

of

reading

frame

the

the

1,893

5'

region This

deduced

HSP70 family

HSP70 family

but

HS2 cDNA might

is

proteins

weight

from

(Table

1).

there

is

clone

has

except

HS2 cDNA nucleotides

has only

be caused

one continuous

by

open

at ATG and extending

capable

open reading

differed

the

starting

DNA

HS2 cDNA clone

of the

molecular

from this

the

genomic

of

nucleotides

a predicted

the

Partial

region

gene

codon TAG, which

with

sequence

in

of

to the EcoRI 6kb

shown).

(GE6) of

stretches

cDNA cloning.

termination

protein

for

mismatched

of

DNA (not

6kb fragment

match

RESEARCH COMMUNICATIONS

band corresponding

genomic

70bp sequence This

I).

mouse

EcoRI

a perfect

an about

a strong

with the

AND BIOPHYSICAL

of coding

630 amino-acid

of 69,645.

frame

We have

The amino

acid

good fit

with

had a very

any previously

sequenced

referred

to

to

mouse

this

gene as

hsc70t.

To examine

whether

gene,

hsc70t

analyzed

total

by the

region

unique

specifically adult

rat

cellular slot-blot

and mouse contain

analyzed

isolated

Figure

by Northern

of pachytene

TABLE 1.

Similarity

hsp70

gene

hsc70 hsp68

(hsc71) (MHS243)*

abundant

levels

11,

10,

from mouse Ltk-

populations

reports

blot cells,

tissues

with

the

untranslated

spermatocytes

3'

have shown that of the

(Fig.

and early

mRNA was

2B).

cells,

(reference) (8)

72.7

72.9

(13)

HSP70.2

71.1

(11)

* A cloned gene encodes only

418

46

carboxyl

terminal

RNA was

and enriched

spermatids.

of hsc70t

of

related

of the deduced amino acid sequence among mouse hsp70 genes

% similarity

were

testes

transcript

of the hsc70t

Ltk-

the

mRNA expressed

hsc70t

hybridization heat-shocked

of

various

Expression

12).

expression

from

2A shows

Recent

testis.

to the hsp70 gene (9, also

RNA isolated hybridization

probe. in the

a tissue-specific

amino acids.

Vol.

166,

No. 1, 1990

BIOCHEMICAL

12

AND BIOPHYSICAL

3

4

1

RESEARCH COMMUNICATIONS

2

3

4

1 2 3

.2.7kb

4 5 6

Fis.

2. hsp70-related gene transcripts in normal mouse tissues, enriched populations of spermatogenic cells and mouse L cells. A: Total RNA (1OHg each) were blotted on the filter and hybridized to a probe derived from the 3' untranslated region of the hsc70t. 1; heart, 2; brain, 3; liver, 4; kidney, 5; IO; pancreas, 6; spleen, 7; lung, 8; stomach, 9; intestine, ovary, 11; epididymis, 12; testis. B: Poly(A)%NA (lug each) denatured agarose gel were electrophoresed in a 1.0% processed for Northern blot hybridization analysis with 39;:

labelled probe derived from the coding region of the hsc70t. lane 1; Ltk- cells, lane 2; heat shocked Ltk- cells, lane 3; spermatids enriched by elutriation, lane 4; spermatocytes

enriched

by elutriation.

C: The Northern

hybridized to the probe derived of the hsc70t.

A probe major

(Ban1

1500bp)

heat-inducible

was present cells,

transcript

spermatocyte observed

induced of

and a less

about

2.7kb

in the early

from

pattern

with

of HS2 as a probe,

the

the

transcript

of

In the faint

spermatocytes

Ltk-

about

cells.

2.7kb

spermatocyte

2.7kb

could

contaminate

3'

population,

fraction

of 47

than

were

in

this

the

the

has been

with

(9,

When the

region not

fragment

recognized

expression

probe

in

of

spermatids

of transcript

the (Fig.

detected seen in

multi-nucleated

spermatocytes

pMHS213,

13).

early

unique

was

in the pachytene

expression

abundant

The amount from

transcript

untranslated

transcripts

was observed

be derived the

the

which

In male germ

This

L-cell

However,

transcript.

cells.

of

two

transcript,

were hybridized

a heat-shocked

was hybridized

RNA from

Ltk-

region

HS2 detected

abundant

population

A similar

isolated

same to B was

of

was observed.

spermatid

(BarnHI-EcoRI)

which

region

when RNAs from the same sources

same filter

very

codig

and noninduced

population.

a cDNA clone

2C).

the

transcripts

in both

more abundant

from

filter

from the 3' untranslated

enriched

a the

spermatids by

the

Vol.

166, No. 1, 1990

elutriation

BIOCHEMICAL

method.

family

is

AND BIOPHYSICAL

We believe

specifically

that

this

expressed

RESEARCH COMMUNICATIONS

member of

in

the

postmeiotic

hsp70

gene

phases

of

spermatogenesis. Another

member of the hsp70 gene family

germ cell about

differentiation

2.7kb

abundant in

in

length

in meiotic

postmeiotic

protein,

P70,

spermatocytes protein

stages is

shown

sequentially

haploid

of

for

two

Further

localization the

hsc70t

gene transcription

spermatids

is

most

in abundance

mouse HSP70-like in

(14,

pachytene The P70

15).

by the HSP70.2 gene.

regulated

differentiation.

a transcript

synthesized

early

testicular

however,

A novel

be primarily in

yields

and decreases

spermatogenesis. to

also

during

transcript,

spermatocytes

and slightly

expression

cellular

of

HSP70.2,

This

(11).

prophase

may be encoded

Thus,

clone

of mouse,

expressed

members

of

during

the

studies

of the gene will

will

product

the

mouse

gene male

be needed

during

facilitate

hsp70

germ

in the male germ cell

is cell

to determine

spermiogenesis. analyses

family

of

the

The DNA

mechanisms

of

lineage.

Acknowledqments: We thank Dr. A. Matushiro for generously providing a mouse genomic library, Dr. T. Miyake for his interest in and support of this study and Ms. M. Hirayama-Yamanouchi for excellent technical We are also grateful to Dr. R. P. Erickson for reading assistance. A part of this study was entrusted to Mitsubishi the manuscript. Kasei Institute of Life Sciences by the Science and Technology Agency using the Special Coordination Funds for Promoting Science and Technology. References Biophys. Biochem. Res. 1. Fujimoto, H. and Erickson, R. P. (1982) Commun. 108, 1369-1375. R. J. and Hecht, N. B. (1983) Dev. Biol. 2. Kleene, K. C., Distel, 98, 455-464. 3. Fujimoto, H., Erickson, R. P., Quinto, M. and Rosenberg, M. P. (1984) Biosci. Rep. 4, 1037-1044. 4. Dudley, K., Potter, J., Lyon, M. F. and Willison, K. R. (1984) Nucleic Acids Res. 7, 179-192. 5. Meistrich, M. L., Trostle, P. K., Frapart, M. and Erickson, R. P. (1977) Dev. Biol. 60, 428-441. E. F. and Sambrook, J. (1982) Molecular 6. Maniatis, T., Fristsch, Cold Spring Harbor Laboratory, Cloning: A Laboratory Manual, Cold Spring Harbor. 7. Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 48

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1, 1990

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10. Krawczyk, Z., Wisniewski, J. and Biesiada, E. (1987) Mol. Rep. 12, 27-34. II. Zakeri, Z. F., Wolgemuth, d. J. and Hunt, C. R. (1988) Cell. Biol. 8, 2925-2932. M. (1989) J. Cell Sol. 12. Krawczyk, Z., Mali, P. and Parvinen,

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49

Rockett,

D. L.

and