1. Gene Organization, Sequences, and Induction 5
protein in higher cells which appears to conespond most closely to the Dros-ophila hsp83 is about 90 kilodaltons in size. In HeLa cells there are three heat shock protein species in the molecular mass range of 90 to 110 kilodaltons (Welch and Feramisco, J982; Welch et al., 1982). Whether these three polypep-tides are structurally related to each other or originate from one or several genes is not yet known.
The protein products of the first and second groups, hsp 70, 68, 27, 26, 23, and 22, are all found predominantly within the nucleus during a heat shock (Arrigo er al., 1980; Velazquez et al., 1980). Upon return to normal tem-peratures, these proteins tend to migrate back to the cytoplasm , where the small hsp's, at least, become associated into ribonucleoprotein complexes (Arrigo et al., 1980; Arrigo and Ahmad-Zadeh , 1981).
The Drosophila hsp genes have nearly all been sequenced in their coding and flanking sequences, and some regions of homology have been noted at their 5' ends. Are these homologies part of a common signal sequence present upstream of these genes which permits regulation of their apparent coordinate induction?
In order to study the transcriptional induction of Drosophila heat shock genes, monkey COS cells were transfected with hsp70 genes cloned into vectors con-taining a Simian virus 40 (SV40) origin of replication. These Drosophila hsp70 gene constructs proved to be stress inducible with heat shock, and, by testing a series of 5' deletion mutants of the hsp70 gene, it was possible to define a sequence necessary for efficient heat induction under these experimental condi-tions (Mirault et aL, 1982; Pelham, 1982). Having observed an apparently imperfect pa.lindromic consensus sequence in front of most of the hsp genes, Pelham and Bienz (1982) synthesized this particular sequence and, by placing it in front of the Herpes thymidine kinase gene in an appropriate vector, were able to render this gene heat inducible in both monkey cells and Xenopus oocytes.
Further investigations in Cambridge and Geneva with the same kind of system are now in progress with other Drosophila hsp genes.
Fo!Jowing the initial observations of Sirotkin and Davidson (1982), several laboratories have now reported tha,t some of the hsp genes are also expressed normally at various times during development under nonstress conditions. Tbe mechanisms of control involved in the developmental expression of the-se genes can now be investigated by transformation of Drosophila, mediated by the P element, as described by Rubin and Spradling (1982).
II. ORGANIZATION AND SEQUENCES A. Heat Shock Protein 70 and Related Genes I. The hsp70 Genes
Following heat shock, mRNA from D. melanogaster tissue culture cells sedi-ments into two major peaks of 20 S and 12 S, respectively (McKenzie and
Meselson, 1977; Spradling et al. , 1977; Mirault et al .• 1978; Moran et al., 1978). The most abundant of the heat shock mRNA's, coding for hsp70, is found in the 20 Speak. It hybridizes in situ to two heat shock puff sites, 87A and 87C on chromosome 3R, indicating the existence of multiple genes encoding hsp70 (Henikoff and Meselson , 1977; Spradling et al., 1977). Genomic clones bearing these hsp70 genes were isolated in several laboratories independently and have been analyzed extensively in order to decipher their organization.
The first heat shock clones to be isolated from D. melanogaster contained an unusual sec of tandemly repeated sequences derived from l.ocus 87C, the so called cx.(3 repeats (Lis et al.. 1978). At this particular location, the transcription of these sequences is heat inducible, but they are also found dispersed at the chromocenter, where they appear not to be stress induced (Lis et al. . 198 l).
Similar sequences are also present, though not transcribed, at the chromocenter of other Drosophila species, such as the sibling species D. simulans (Livak et al. . 1978). It seems likely, therefore, that the af3 repeats have been brought to 87C as a result of recent evolutionary events in D. melanogasrer. They do not appear co code for any protein product and their role at this locus remains obscure.
ln addition to the af3 repeats, a variant unit called <X."'f has also been observed interspersed within af3 clusters at 87C. The af3 sequences are transcribed upon heat shock into RNA of three size classes, the largest of which is 3 kb long and corresponds to aj3a. Interestingly, "Y sequences are found at both 87C and 87A, the two sites expected to bear hsp70 genes. Lis et al. (1978) therefore speculated that the -y element contained sequences involved in the beat induction of both aj3 and hsp70 genes and this prediction turned out to be correct.
In the meantime, a number of D. melanogaster clones containing hsp70 gene sequences were isolated and characterized in different laboratories (Livak et al., 1978; Schedl et al. , 1978; Artavanis-Tsakonas el al. , 1979; Craig et al., 1979;
Moran et al. , 1979). These genes were derived from both loci 87A and 87C. To each of these two chromosomal sites there con·esponds a characteristic gene variant. The first structural outline of the hsp70 multiple gene organizatien emerged from the comparison of two clones, one containing a single-gene copy from 87 A, and the other, two genes in a tandem repeat from 87C (Moran et al., 1979; Artavanis-Tsakonas er al. , 1979). Each individual hsp70 gene is organized as a basic, conserved unit Z , consisting of a 2. 2-kb segment encocling hsp70 mRNA, Zc (Z coding), and a 0.35-kb region, Znc (Z noncoding), present at the 5' end of Zc. No introns have been observed in these genes. Additional regions of sequence similarity, the so-called X elements, form a complex pattern of DNA homologies present upstream of the hsp70 genes at both 87 A and 87C (Moran et al., 1979). Several clones derived from 87C contained
a.13
sequences in the close vicinity of an hsp70 gene variant (Livak et al. , 1978; Craig et al., 1979). This was the first direct evidence that these two gene types, cxj3 and bsp70, are closely linked physically at this locus.(
I. Gene Organization, Sequences, and Induction 7
The global organization of the Drosophila multiple hsp70 genes at both 87 A and 87C was deduced by a combination of detailed genetic and molecular analy-ses of these two loci. The characterization of a series of overlapping deficiencies (deletions) covering one or the other locus, or both, has allowed the localization of the hsp70 genes to the two cytological bands 87A7 and 87Cl (Ish-Horowicz et al. , 1977, 1979a,b ). Embryos lacking these two bands fail to synthesize hsp70 following heat shock, whereas this protein is made in embryos retaining either 87A7 and/or 87Cl. The restriction map of the hsp70 gene sequences was exam-ined directly in the genome of the mutant strains that lacked either the 87 A 7 or the 87Cl band, or both (Holmgren et al., 1979; Ish-Horowicz et al., 1979b; Ish-Horowicz and Pinchin, 1980). Each locus was found to contain multiple copies of its own particular hsp70 gene variant, identified by characteristic restriction sites. The overall arrangement of the hsp 70 genes found at 87 A 7 and 87C 1 of most D. melanogaster strains is displayed in Fig. 1.
The locus 87 A 7 contains two genes in opposite polarity (Mirault et al., 1979;
Ish-Horowicz and Pinchin, 1980), as found in clone 122 (Goldschmidt-Cler-mont, 1980). Polymorphic variants of this gene arrangement have been isolated, which differ by the deletion or insertion of large DNA stretches. For example, one clone contains the distal hsp70 gene of 87A7 and a copialike dispersed, middle repetitive sequence located just upstream of the heat shock gene (Ar-tavanis-Tsakonas et al., 1979); two other clone isolates lack most of the spacer DNA and the 5' half of the proximal gene (Goldschmidt-Clermont, 1980; Udvar-dy et al., 1982). The latter authors speculated that incorrect excision of a trans-posable element could have generated these apparent deletions. Another deletion mutant has a truncated proximal gene at 87 A 7 lacking its 3' moiety; in-terestingly, embryos homozygous for this mutation and deleted of the 87C locus synthesize a new hsp70-related heat shock protein of 40 kilodaltons, in addition to the normal hsp70 encoded at 87 A (Caggese et al., 1979; Burke and Ish-Horowicz, 1982). This indicates that both hsp70 genes at 87 A 7 are activated by heat shock.
The organization of the hsp70 genes at the 87Cl locus is more complex than that at 87A7 (see Fig. 1). The hsp70 genes are found in two domains, a single proximal gene separated by about 40 kb of DNA, which contains clusters of al3 sequences, from two tandem gene copies present in the distal part of locus 87Cl (lsh-Horowicz and Pinchin, 1980). In some strains and in the Kc tissue culture cell line there are three tandem gene copies. The total number of hsp70 genes at this locus can vary between three and five copies according to genotype and fly stock (Craig et al., 1979; Ish-Horowicz et al., 1979b; Mirault et al., 1979).
Similarly, the number of al3 and a-y repeat units has also been reported to vary, from 8 to 14 and from 5 to 8, respectively (Lis et al., 1978, 1981). It was thus suggested that unequal crossing-over events had generated these variations and that the tandem duplication or triplication of hsp70 genes may have arisen by the same mechanism. Such events may well have been favored by the variable
hsp 70 Zc
87A7
hip 70 ~c zext hsp 70
Zc Zn~Znc Zc ~ b p
87C1 Zex l '···--···~---... ... a. .,_ ~ r ... Xbk. ... ~>:.~~.~L hsp70
...
hsp 70Znd : : Xa Xb l[Znc k XcZnc le
!. •
r
I "' I P I "- I ll I o. I P Hi-: !..../1-ll--D!HH.. ... [HD••iil••- .
hsp 68
950
hSp 27 ~sp 23 ge ne 1 hsp 26 hsp 22 gene 2 gene 3
67 B I >
c=i-c:::::::>--hsp 83
63BC ~N
Fig. I. The arrangement of the Drosophila mela11ogastl!r heat shock genes. Those regions complementary 10 heat shock mRNA are indicated by black arrows, the orientation of which gives the direction of mmscription of each heat shock gene. The top two maps ponray the organization of the hsp70 gene family at the cytological loci 87A7 and 87C i, respec!.ively, and rakes inro account the sequence data from Hackett and Lis (1981), Karch et al. (1981), and Mason et al. (1982). The left-hand pan of these two maps is proximal to the centromere of chromosome 3R. As described in the text, the 2.2-kb Z,, element is complementary to hsp70 mRNA. The 0.35-kb Z,,., regions (hatched boxes) are approximately 98% conserved at each locus and 90% homologous between the two loci (Mason /JI al .. 1982; Torok cl al. , 1982). Additional areas of homology (X., Xb, X0 , and Zcxol are found 10 be both very AT rich and well conserved. X, and Xb were first discovered by heteroduplex analysis (Moran et al .. 1979; Goldschmidt-Clem1ont, 1980). X0 and the 0. 15-kb Zcxi (Z extension;
cross-hatched boxes) were found by sequence daca comparisons (Mason er al .. 1982). The 'Y element consists of the first 64 bp of Z.,. the entire Znc and Zc .. elements, plus a small additional region (s{ipplcd box; Lis et al .. 1978; Hackett and Lis, 1981; Mason et al. , i 982). The o:.13, and 'Y elements are arranged as either al3 or <l'Y candem arrays in the approximately 40-kb spacer which separates the single proximal hsp70 gene from two distal hsp70 gene copies at locus 87C i . The precise order of these different elements is unknown except for the aj3 and CX"f repeats which have been anaiyzed in cloned DNA's (Lis et al., 1978; Holmgren et al. , 1979). The third map. representing the hsp68 gene m locus 95D, was rnken from Holmgren el al. ( 1979, J 98 l). The fourth map gives tbe organization of the 670 locus (Corees l!t al., 1980; Craig and McCarthy, 1980; Voellmy et lll., 1981 ; Sirotkin and Davidson, 1982). The location of the four small hsp genes is indicated by the black boxes and that of the three developmentally regulated genes by open boxes (nomenclature taken from Sirotkin and Davidson, 1982). The map of the hsp83 gene at locus 63BC comes from Holmgren et al. (1979, 1981). The hsp83 gene is the only Drp!;opliila heat shock gene to possess an iotroa.
number of u~ tandem repeats. Interestingly, u~ sequences have not been de-tected at the 87C locus of any other Drosophila species investigated so far.
Instead, the closely related species D. simulans and D. rna11ritiana and the more distantly related D. teisseri and D. yakuba all have two pairs of inverted hsp70 genes which probably represent the ancestral organization that existed at both
1. Gene Organization, Sequences, and Induction 9 87A and 87C before D. melanogaster became separated from the other species (Leigh-Brown and Ish-Horowicz, 1981). Thus, the insertion and propagation of af3 sequences, as well as the tandem duplications of the hsp70 genes, seem to be recent and related evolutionary events at the D. melmzogaster 87CJ locus.
There is another observation that supports this interpretation. The 0.35-kb sequence Znc• which is conserved 5' to all hsp70 genes in D . melanogasrer (Ish-Horowicz et al., 1979b; Mirault et al., 1979), shows extensive homology to the part of the 'Y element which is found adjacent to several
a13
repeats (Lis ec al., 1981). Sequence comparison between the 'Y element and the two hsp70 genes at locus 87Cl revealed that 406 base pairs (bp) of the 'Y unit present at the 5' end ofo.13
repeats shared a near-perfect homology (98%) with the entire znc and the first 64 bp ofZc (Hackett and Lis, 1981; Mason et al .. 1982). These data suggest that this common upstream sequence contains cis-regulatory signals conferring tran-scriptional inducibility to both hsp70 and af3 sequences. The complete element, found immediately adjacent to the proximal hsp70 gene of 87Cl, is therefore thought to be the ancestral sequence which became interspersed among af3 repeats by some unknown mechanism, consequently bringing them under heat shock control (Mason et al., 1982; Torok et al., 1982). The remarkable conser-vation of both 'Y and the Znc element of hsp70 at 87Cl suggests that the dispersal of 'Y at this locus must be a recent event, since Znc at 87 A 7 has diverged significantly from both Znc and 'Y at 87Cl (Hackett and Lis, 1981; Karch et al., 1981). The apparent ancestry of the 87Cl locus in D. melanogaster provides a striking example of rapid gene evolution, in that some of these genes have come under the transcriptional control of others.Sequence comparison between an hsp70 gene from locus 87Cl (lngolia et al., 1980) and its 87A7 gene variant (Torok and Karch, 1980; Karch et al., J98J) indicates a 4.2% nucleotide divergence within the mRNA coding region Zc, with complete conservation of an open, uninterrupted reading frame. Most base sub-stitutions occur in the third codon position, and 0ut of a total of 643 amino acids, there are only 26 substitutions and 2 insertions; this would correspond to a 2. 7%
divergence of the amino acid sequence (Karch et al., 1981). The start of tran-scription, which defines the boundary between zc and znc• has been localized 250 nucleotides upstream from the putative initiation codon by S 1 mapping (Torok and Karch, 1981). Most sequence divergence is located within the 5'-untranslated regions, as first reported by Holmgren et al. (1979), and in the 3'-noncoding ends of the two mRNA variants, which are totally nonhomologous (Karch et al., 1981; Torok et al., 1982). The Znc sequences upstream of the 87A7 and 87Cl gene variants display 15% divergence. In contrast, however, at each locus both Znc and Zc sequences are remarkably well conserved among the copies of each gene variant (Karch et al., 1981; Mason et al., 1982).
A structural comparison of the hsp70 genes in several species related to D.
melanogaster led to the conclusion that these genes have apparently not evolved independently from one another (Leigh-Brown and lsh-Horowicz, 198 l). These
authors suggested both intralocus and, more infrequently, interlocus gene con-version events to exp.lain the apparently concerted evolution of the hsp70 genes at these two loci. Comparison between the extragenic sequences upstream of the hsp 70 genes at both 87 A 7 and 87C 1 revealed a complex pattern of sequence homology (Moran et al. , 1979; Goldschmidt-Clermont, 1980; Hackett and Lis, 198 J; Karch et al. . 1981; Mason et al. , 1982) . The relative an-angement of the different X elements is given in Fig. 1 . The origin and possible function of these sequences remain obscure, but the pattern of homology observed at the DNA level suggests that these elements could have arisen as a resuJt of interlocus gene conversi.on and/or transposition evenrs (Mason et al., 1982; Torok et al. , 1982).
X elements do not appear to be essential for the heat shock induction of hsp70 genes since a mutant Drosophila strain possessing only one hsp70 gene copy, with no flanking X elements, is perfectly viable and responds normally to heat shock (Udvardy et al., 1982). It is possible, however, that X elements are involved in more general mechanisms associated with some selective advantage, such as recombination, DNA replication, or DNA transposition. In this respect, we found that X sequences are not restricted to the 87A7 and 87Cl heat shock loci but are found at many other sites within the D. melanogaster genome (Lis et al., 1981; M.-E. Mirault, unpublished observations).
Unexpectedly, however, hsp70 gene probes were found to hybridize in situ not only to the chromosomal subdivisions 87 A and 87C, but also to the heat shock puff locus 95D and to site 87D, subdivisions 10, 11, and 12, which is not known to be involved in the heat shock response (Holmgren et al., 1979). Both sites possess genes structurally related to hsp70, the former locus containing the gene encoding hsp68, and the latter, a he.at shock cognare gene (Craig er al., 1983; see Section 11,A ,3).
2. The hsp68 Gene
Two DNA clones have been described that bear hsp68 gene sequences, as shown by hybrid-arrested translation studies. One clone contains most of the gene but is missing the 5' end (Holmgren et al., 1979), whereas the other clone contains a complete gene copy (Holmgren et al., 1981). Hybridization of the latter clone with wbole-cell heat shock RNA protected a 2.1-kb fragment against both Sl and exonuclease VII digestion , thus indicating the absence of introns within this gene. Heteroduplexes between hsp68 and hsp70 genes revealed the existence of a strong cross-homology, but duplex melting studies indicated ap-proximately 15% sequence divergence (Holmgren er al. , 1979). This homology suggests that both genes may have evolved from a common ancestral gene and that the 68- and 70-kilodalton heat shock proteins could have a similar or a shared function(s). It is perhaps no coincidence that embryos lacking the 87A7 and 87Cl loci synthesize significantly more hsp68 than do normal embryos
'
(
(
I. Gene Organization, Sequences, and Induction 11
following heat shock, possibly as a result of dosage compensation for the lack of hsp70 (lsh-Horowicz et al., 1977).
3. The Heat Shock Cognate Genes
The Drosophila genome contains several hsp70-related genes, the transcrip-tion of which is not heat shock-inducible but regulated during development (Craig er al., 1982, 1983; lngolia and Craig, I 982a). The first of these so-caJled heat shock cognates, Hscl, was isolated from the two sibling species D. melano-gaster and D. simulans and was found to be located at cytological locus 70C on chromosome 3 (lngolia and Craig, I 982a). Since then , rwo other heat shock cognate genes, Hsc2 and Hsc4, have also been isolated from D. melanogaster and found to hybridize in situ to the same chromosome at cytological loci 87D and 88E, respectively (Craig et al. , 1983). Sequence comparison of about one-third of the protein coding regions from aU three cognate genes with an hsp70 gene showed approximately 76% homology at the DNA level and about 78%
homology at the predicted amino acid level. Comparison of the Hscl gene DNA sequence from the two Drosophila sibling species i·ndicated, after optimal align-ment, only a few mismatches.
As shown by hybridization selection and translation experiments, RNA ho-mologous to Hsc4 encodes a protein of approximately 70 kilodaltons with a similar, though distinguishable, electrophoretic mobility to that of hsp70. Due ro the variable lengths of the Hsc 51 leader sequences, it was possible to distinguish specific transcripts from each gene during development by means of cDNA primer extension experiments. Hscl and Hsc2 transcripts were undetectable in both embryonic and larval RNA preparations but were abundant in adult fly preparations. In contrast, however, Hsc4 mRNA was found to be equally abun-dant, at lev.els comparable to actin mRNA, at all three developmental stages. The relative abundance of the Hsc transcripts is not increased by heat shock.
Hsc4 contains no intron in the sequenced region encoding the first 101 amino acids, at least, but this is not the case for the other two cognates. Hscl is interrupted by a I. 7-kb intron inserted into the codon specifying ami.no acid 66, whereas Hsc2 has a 0.65-kb introo present in the codon defining amino acid 55.
The sequences bordering the introns in Hscl and Hsc2 agree with the consensus splice site sequences in Drosophila and other eukaryotes (Craig et al., 1983).
4. The Yeast hsp70-Related Genes
4. The Yeast hsp70-Related Genes