characterization of a g-protein β-subunit gene from the nematode caenorhabditis elegans

J. Mol. Biol. (1990) 213, 17-26

Characterization of a G-Protein p-Subunit Gene from the Nematode Caenorhabditis elegans

Loesje van der V o o m , M a r t i j n G e b b i n k , R o n a l d H . A . P las terk a n d H i d d e L. Ploegh~"

The Netherlands Cancer Institute Plesmanlaan 121

1066 C X Amsterdam The Netherlands

(Received 13 September 1989; accepted 19 December 1989)

The gene encoding the fl-subunit of guanine nucleotide binding regulatory proteins (G-proteins) has been cloned from the nematode Caenorhabditis elegans. The predicted 340 amino acid sequence matches the highly conserved amino acid sequences of previously isolated G-protein fl-subunits from mammals and Drosophila. The coding region of the C. elegans fl-subunit gene, which has been mapped to the C. elegans chromosome II, is interrupted by eight introns. Southern analysis indicates that C. elegans has only one fl-subunit gene. A 2.8 kb (1 kb = l03 bases or base-pairs) transcript derived from this gene could be detected.

1. Introduction

Guanine nucleotide regulatory proteins (G-proteins) are involved in the transduction mechanism of a variety of signalling systems. Members of this family of structurally homologous proteins serve to transduce stimulatory or inhibitory signals to intra- cellular effectors, in response to activation of a specific type of receptor (Gilman, 1987).

G-proteins, which are apparently present in every cell type of higher eukaryotes, are heterotrimeric proteins consisting of an a (39 to 52 kDa), fl (35 to 36 kDa) and T (8 to 10 kDa) subunit. Signal transduction via G-proteins involves GDP-GTP exchange, which occurs on the a-subunit in response to receptor activation. Upon GTP-binding, the a and fly elements are thought to dissociate. In some mammalian signalling pathways, an "activated" a-GTP subunit has been shown to interact with an effector, such as adenylate cyclase or cGMP- phosphodiesterase (Stryer & Bourne, 1986). On the other hand, it has also been suggested that the fl~ element interacts directly with an effector. Such a model has been proposed for retinal rod phospho- lipase A: (Jelsema & Axelrod, 1987), atrial potas- sium channels activated by muscarinie agonists (Logothetis et al., 1987) and inhibition of adenylate cyclase (Katada el al., 1984). Although these

t Author to whom correspondence should be sent at: Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

0022-2836/90/090017-10 $03.00/0 17

observations in mammals are still controversial, genetic data from the mating response pathway in yeast strongly suggest that the dissociated fl? element, and not the a, initiates the pheromone response (Whiteway et al., 1989). G-Protein action is terminated by hydrolysis of GTP to GDP, followed by reassociation of a and fl? subunits to form the inactive, GDP-occupied afl~ heterotrimer.

Biochemical characterization and molecular cloning studies have thus far demonstrated the presence of at least nine distinct but highly similar a-subunits (Kaziro et al., 1988) and three ?-subunits (Ovchinnikov et al., 1985; Gautam et al., 1989) in mammals, eDNA clones corresponding to three different mammalian fl-subunits (ill, f12 and f13) have been isolated and sequenced (Sugimoto et al., 1985; Fong et al., 1987; Levine et al., 1989) and evidence for the existence of more fl-subunit genes is accumulating (see Discussion).

The fl-subunit is a 340 amino acid protein which is largely comprised of repetitive homologous elements, arranged in tandem (Fong et al., 1986). The ill, f12 and fla subunits are structurally homologous (80 to 90% similarity at the amino acid level), but encoded by separate genes. I t is not known whether the rather subtle differences in structure between these three fl-subunits reflect functional differences.

Genetic manipulation of G-protein subunits will likely be required for a complete understanding of their functions in vivo. In this light it would be helpful to have cells or organisms lacking the expression of a specific G-protein subunit. The high

© 1990 Academic Press Limited

18 L. van der Voorn et al.

degree of conservation of G-protein subunits and the evidence t ha t G-proteins exist in invertebrates , p rompted us to search for homologous genes in the nematode Caenorhabditis elegans. Genetic analysis of C. elegans is well-developed and m a n y mutan t s are known, including some with abnormali t ies in signal t ransduct ion processes (Wood, 1988), suggesting the possibility tha t C. elegans m a y be a useful sys tem to s tudy the functions of G-proteins in vivo.

Here we describe the cloning of a G-protein fl-subunit gene f rom C. elegans, and repor t the in t ron-exon organization of a fl-subunit gene. The predicted amino acid sequence is compared to known fl-subunits in mammals . We have no evidence for the presence of more than a single fl-subunit gene in C. elegans. Concordantly, only a single fl-messenger RNA is detected in Nor thern blots of C. elegans RNA.

2. Materials and Methods

(a) Nematodes

The wild-type strain of C. elegans vat. Bristol (N2) was used in these studies.

The methods used for cultivation of C. elegans have been described by Brenner (1974). Bacteria-free C. elegan8 of heterogeneous age were prepared as outlined by Sulston & Brenner (1974). Packed C. elegans were frozen at -80°C until use.

(b) Immunoblotting

Frozen worms were homogenized in cold 10mM- Tris" HCI (pH 8'0), 2 mM-MgCl2, using a Polytron mixer set at maximum speed. After freeze-thawing 3 times, the homogenate was centrifuged at 25 g at 4°C for 15 min. Subsequently, the supernatant was centrifuged at 12,000 g at 4°C for 15 min. The resulting pellet was resuspended in 10 mm-Tris" HCl (pH 8"0), 2 mM-MgCI 2. The protein content was determined by the method of Bradford (1976).

Membrane proteins were treated with SDS and N-ethylmaleimide as described by Sternweis & Robi- shaw (1984). After separation by SDS/PAGE (Laemmli, 1970) on a 12% (w/v) gel, immunoblotting was performed according to the method described by Towbin (1979). The primary antibody used is a rabbit anti-human brain G-protein polyclonal antiserum (to be described else- where). As a secondary antibody, alkaline phosphatase- conjugated swine anti-rabbit immunoglobulins (Dakopatts) were used. Alkaline phosphatase activity was detected by staining the blot with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Promega).

(c) Library screening

A A2001 library, constructed from a Sau3A partial digest of C. elegan8 genomic DNA, was kindly provided by J. Sulston and A. Coulson. Screening of the library was performed by the plaque hybridization technique described by Benton & Davis (1977). DNA probes were labeled with [a-S2P]dCTP (Amersham), using random oligonucleotides as primer (Feinberg & Vogelstein, 1983) (Boehringer kit). For low stringency hybridizations, baked filters were prehybridized for 1 h at 37°C in 30 % (v/v) formamide, 0"9M-NaC1, 0"09M-trisodium citrate

(pH7-0), 5x Denhardt's solution, 0"5% (w/v) SDS, 0"01% (w/v) denatured herring sperm DNA. The denatured probe was added to this solution and hybridized overnight at 37°C. Filters were washed in 15 mM-NaCl, l'5mM-trisodium citrate (pH7"0), 0"1% SDS at 37°C. High stringency hybridizations were performed in the same buffer, but at 48°C.

Isolation of C. elegans genomic DNA was performed by the proteinase K/SDS method as described by Emmons et a/. (1979). General nucleic acid procedures were performed according to Maniatis et al. (1982)+

(d) Isolation of RNA

Frozen, packed worms (1 ml) were ground to a powder in liquid nitrogen using a mortar and pestle. Ice-cold homogenization buffer (1% (w/v) SDS, 5% (v/v) phenol, 50 mM-Tris'HC1 (pH 8"5), 50 mM-EDTA (pH 8"0)} was added (3 ml) and the worms were homogenized on ice using a Polytron mixer. The homogenate was extracted 3 times by adding 1 vol. phenol/chtoroform/isoamyl alcohol (25:24:1, by vol.) followed by gentle shaking for 5 min and centrifugation for 5 min at 1500 g at room tempera- ture. After precipitation with ethanol, the pellet was resuspended in 2 ml of l0 mM-EDTA (pH 8-0). An equal volume of 5 M-LiC1, 10 mM-EDTA (pH 8"0) was added and the RNA was precipitated for 2 h on ice. The solution was centrifuged for 30 min at 25,000 revs/min in a SW41 rotor (Beckman) at 4°C. The pellet was washed with 96% (v/v) ethanol (-20°C) and resuspended in water.

Poly(A) + RNA was isolated according to Maniatis el al. (1982) with the modification that the washing step with loading buffer containing 0"l M-NaCl was omitted from the procedure.

Total and poly(A) + RNAs were separated on 1% agarose/formaldehyde gels (Meinkoth & Wahl, 1984) and transferred to Hybond N membranes (Amersham) (Thomas, 1980). Baked filters were prehybridized at 42°C for 1 h in 40% (v/v) formamide, 50 mM-NaPi (pH 7-4), 0"9 M-NaCI, 0"l~o (w/v) SDS, 5x Denhardt's solution, 10% (w/v) dextran sulfate, 5mM-EDTA, 0"01% (w/v) denatured herring sperm DNA. Hybridizations were conducted overnight at 42°C in the same solution containing denatured [32P]DNA probe. Blots were washed twice with 0"45 M-NaCI, 45 mM-trisodium citrate (pH 7"0), 0"1% (w/v) SDS and once with 45 mM-NaC1, 4"5 mM-trisodium citrate (pH 7"0), 0"l ~o SDS at 58°C.

(e) Nucleotide sequencing

DNA sequence determination was performed by the dideoxy chain termination method of Sanger et al. (1977), using the Pharmacia T7 sequencing kit. DNA sequencing was performed with single-stranded templates in M13mp vectors (Messing, 1984). Oligonucleotide primers were synthesized on a New Brunswick Biosearch DNA synthesizer.

Computer analyses were performed using the GCG programs (Devereux et al., 1984).

(f) RNaze protection

RNase protection experiments were performed as described (Melton et al., 1984). The expression vector pGEM3zf(-) (Promega) was used to synthesize a [a2P]UTP (Amersham)-labeled antisense RNA probe.

C. elegans G-Protein fl-Subunit Gene 19

Protected fragments were separated on 6% (w/v) acrylamide/8 M-urea gels.

3. Resu l t s

(~) Immunoblotting

C. elegans membrane fractions were analyzed by immunoblotting for the presence of G-protein subunits (Fig. 1). Although none of the sera tested detected polypeptides in C. elegans extracts in the molecular weight range reported for a-subunits from other species (results not shown), serum H1, directed against purified human brain G-proteins, recognizes a clear 36kDa fl-polypeptide in C. elegans membranes. As compared to the control lane, where 1 ~g of purified human brain G-protein was loaded, the amount of immunoreactivity in C. elegans membranes suggests that the fl-subunit is a quite abundant protein in C. elegans and that it must be structurally similar to the human fl-subunit.

(b) Cloning and sequencing

To isolate the G-protein fl-subunit gene from C. elegans, approximately 6 × 104 plaques (10 genome equivalents) of the C. elegans 22001 genomic library were screened. As a probe we used a 1.4 kb~f bovine transducin fl-subunit cDNA, fi l l2 (Fong et al., 1986) under low stringency conditions. The DNA of 12 positive clones was analyzed by restriction enzyme digests and subsequent Southern blotting. This showed that all positive clones were overlapping and from the same genomic region. Sequencing of a strongly hybridizing 0"3 kb BamHI-BglII fragment, present in all positive clones, revealed G-protein fl-subunit-like sequences.

From one of these positive clones (fl215) a 7 kb SacI fragment that seemed to contain all hybridizing sequences was subcloned in pUC18 (fl215pS1). After further subcloning of suitable fragments in

Abbreviations used: kb, 10 ~ bases or base-pairs; bp, base-pair(s).

1 2 3 4

~ m

O -

Figure 1. Analysis of G-protein subunits by immunoblotting. A total of 100 #g of C. elegan8 membrane proteins (lanes 2 and 4) were analyzed by immunoblotting, as described in Materials and Methods. The antisera used were: H1, a polyclonal rabbit antiserum, raised against purified human brain G-proteins (lanes I and 2) and normal rabbit serum (lanes 3 and 4). Separate strips from the same nitrocellulose filter are shown.


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Figure 2. (a) Restriction map and sequencing strategy for the C. elegans G-protein fl-subunit gene as present in clone fl215S1. Restriction sites: A, AccI; B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; S, SacI; Sin, S~mI; X, XbaI; N, NarI (only the NarI site used for preparation of probe B (see (c)) is indicated, but additional NarI sites are present in clone fl)A5S1). Arrows depict separate DNA sequence determinations. (b) Genomic organization of the C. elegans fl-subunit gene. Boxes represent exons. Filled boxes represent coding regions, whereas the open boxes represent non-coding regions. Broken lines flanking a boxed region indicate sequences that are transcribed as concluded from Northern blot hybridizations (results not shown), but where no information about intron/exon organization is available. (c) Origin of probes used in Southern and Northern hybridization or in RNase protection experiments (see Figs 5 and 6).

M13mpl8 and mpl9, the complete nucleotide sequence of the 7 kb SaeI fragment was determined on both strands.

A map of restriction sites present in clone fl~lSS1 and the orientation of DNA fragments used for sequencing is presented in Figure 2.

(c) Structure of the C. elegans G-protein fl-subunit gene

The nucleotide and the predicted amino acid sequence of the C. elegans G-protein fl-subunit are shown in Figure 3. Exon sequences were identified based on homology with mammalian fl-subunit amino acid sequences and the presence of consensus splice sites (see below). The resulting reading frame specifies a 340 amino acid protein. That we indeed cloned a functional G-protein fl-subunit gene was indicated by (1) the deduced amino acid sequence, which matches the highly conserved amino acid sequences of previously isolated G-protein fl-subunits (Sugimoto et al., 1985; Gao et al., 1987; Yarfitz et al., 1988), and (2) Northern analysis and RNase protection experiments (see below). The coding region of the C. elegans G-protein fl-subunit gene consists of nine exons separated by eight introns, all

flanked by C. elegans splice signals (Wood, 1988). Sequences resembling the transcription signal sequences TATA (Corden et al., 1980) and CAAT (Benoist, 1980) are not found upstream from the initiator methionine. Two in-phase termination codons are found at position -71 and - 117 relative to the start codon. There is no consensus poly- adenylation signal (AATAAA) (Proudfoot & Brownlee, 1976) found within the 1350 nucleotides sequenced downstream from the termination codon. At nucleotide position - 164 relative to the initiator ATG, a consensus C. elegans splice acceptor site is found. To test whether this site is used, we performed RNase protection experiments (Fig. 4). The B a m H I - S m a I fragment (position -780 to +611) derived from fl).15S1 and subcloned in the pGEM3zf{-) vector was used for the synthesis of uniformly [32P]UTP-labeled complementary RNA. Hybridization of this probe with C. elegans total RNA, followed by RNase A and RNase T 1 diges- tion, resulted in the protection of a fragment of 221 bases, which is exactly the size of the exon between the presumed splice acceptor site at position -164 and the next splice donor site at position +57. From this experiment, we conclude that the splice acceptor site at position -164 is used.

C. elegans G-Protein fl-Subunit Gene 2l

T6~TCT~TCTCT~TCTCCATAGGAATAGTGTTCTGAATGTA6AGGTCTAGA~GAG~CTCTGATTG6T6TATCTC~CAGCTCACT6GCACACACACA~ AC6CACACACACACACGCTCAC~CTCAATGTTCTACTTCTTCTACGTCGTCGTC~TCTTCTACTTCTACATCTACTTCTTCTTTCTTCTT6T6CTTCGTC TCA~GGCATTATCTGCATATGC6TGTCTGTTTCTCTG6GCTGTGTGTTGCTTAGTCGAATCGACTGATT6CCTGACACAATCCGCTTATTTTTTCTTTTTTT

CCTTCTAGTCCAGAAC6TTTCGCCGCTTTTTCATTTCCTGTGCCCATTCACATTTTAGACCCAAACACCAGCCATTAA6CCGTCTTGC~TCCTGC~AC GTCACATCAAAGCCATTAATCTTCGATGGCAGTCTGGCTCGGTCAAAATTC~CCTATTTTTGTCTGAGAACTTT6TACAATTTTATTGGTTAAAAAT6CT CGCACCACCTGCTGGAGGGCACCAGATGTCCCCTCGCCCAAAGCAATTAGAATTCTGCATTTAGCAAACAAAAGTT6AATCAATAATTCGCAGATCCAAT GGTA6GA6AGAGAAA6AGTGA~CC~C~CCTTCTTCGCACAGATATATATTAGCTTTTGTCACGTTTT~C~CTCCTACCTG6GAGAGCCCAA6GTATTTGA A~AC6GA6AGCAATATGTTCCCTCAAAACTCAAATACCTGTGCCCCATCATTTCCTTCCTTAATTCCCTCATTTCATTC6ACATTTGTC~TATTCA~TTT CTTTTCTTTCTTTTTTGTCATT6TTTTGTTCTTCCGCGCCACACACACACACACAGACCAACACTTTTACATTTTTTCCCGCCCGCTCCCCTTTTCCATC CC~CTTGGTCTGCGTCTCAAGGTTTGGTTTCTTTCTC6TACAGTGTTCCGCTCTCACGCCAGTTTCTGAATCGATCTATTGATTTTTTGAAAGTTAAAAA TTTTGCTTAAAATTTCTGAAGCTTATGATTTTGGTTTGCAGACCGTCATTCTGTACTTCCTGATCAGCAGCTTG•TCTCACCGTCTTTGCCAAGTTGACG TCGGTCGAG6GGTTTTAATTAAAA~C~TTAGTGATATAGATTTCTTTCTAGTAGGTTTTTCATGTTTT~ATCAAAAACATACCTTTA6TTTATTCTTATT TTATTATCTCACCTATA666ATCCCTAGAGCTAATAATCTACTTCCAAAGTTCCTCTCTAAAGAATTAAATATTTTCCCGTCTA~TTATCGATTTTA6AA TCGAACCTATTTTATTAGATAAGTAAGACCAAAATGTGTTGTTTTTTCAAATATAGAATTCTGGAATCACTTATTTTTAAATTGAAAAAAATCCATTTTT TTCAAATTTTTGATTTTTACATAAATTACTT~ATTACAAT6AAACAATCAATAATTTTCTTCATTTTTT6ATT6GTTTTCGCATTGAATATTCAATTTAA AACTTATTAATTAATTAATTAAATAAAAACAAAAAACATTGCGCGGCAATTAGCAT~TGCTGG C CTACCGTAGTTTCTGCAAACACC6TGACGTCAATAT GCACACTGTT6TTTTTCTTT6ATTTTCTTTCCTTTTTTGACCTTTTTTATCTTATTTTTTTCTTATTTTCTC CACAGAAAATCTCTAAAAGCTTCGAAAA CTTGAATTATTTTAACGATAAATTTAATTCCTTCATGAAAATAATCAAATTCTT~AAAAAATCGATTTTTTCTGCTATTTCCTTCAAACTC ~ ~ ~ ~ ~ ~ ~C

~2~TAT~MT~TT~TT~CA~G~TT~T~.TG~A~C~T~TCGAGTG~MGM~U~A~A~C~LAGA~GA~GAATAGAG~G~G~G~GT~

- 1 ~ AAAC6CG~CA~C~A~CA6GAGCAGCA~CCCCAGCCG~ATCCGTCGA~ACTT~CATC~TA~CATCCT~C~CACG~ACCAC~AC~CAGC~6

fl S E L O Q L" R Q E "A E Q L K S Q" I R 0 ATGA~TT~A~C~A~A~A~CTG~CAG~TG~TCG~AGATTC~GTGAGTAAAT~TGT~TTT~AW~l lwiA~TAT~TT

i 1 AAA~T~T~TT~CA~T~TCTCT~GTTCCAGA~CAT~T~TA~AATT6TTT~AATATGC~AAATATGA~AAATA~A~6~ATTTATATA~TATA~A~A CACATATACACACATACATATACAAATTGTT~T~T~TAAAGCGCTCCCCTTCC~C6ATGCC~CC~CCGCC6CCATC~CG~TTT~CGCCGT ACTTGA~G~GACT~CTGCTGCACT~ATGTGTAT~CACGATGGTCGTCGTCCCCCAGCCGTCGTCGTCTTC~iC~C~C~CTACCCGCCATCGC CGCT~CAAAATATCGATCAGTA~CA6~6TT~TTGCTCCACACACGCA~CCGCGGC6AGCCATT~CCGAGAT6C~d3TC6CCCCT~T6GGGGCACTGA GACGA~AG~GCTG6AGCA6CA6CAG~GCAGC~AGCGAGC~AGGAG~TAGATTGGCTGCCT~TGTGAGA~GTC~CGTCTCC~CTC~GTTCCA CTA~ICCCGGGGCCCC6CC~CCG~TGCTCTC~TTTCTGCCGCTACTGC~GCC~GAGCACGAGAGA~AGA6AG~AGATCTCTCGCAGTCTCTCT TCTGCGCAAGCTCCACTCGTGTCGTAGTGCGCCATTCTCCCTTGAGGACTC~CTGCCGTCGTCGTC6TC6~GTC6~GTCATCGCGTCGCTCACTAGA A~T~TAGCTCC~CTCCGACGAGCACTCACACACACACACAT~T~CGTGTGCGCGAGCACAAAATGT~CGCGGGGTTT~T6~GCTCTC~ T~C~AAAAC~GG~CT~CCT~AGCGA6~A~CAGCAGAGATAG~TACACTG~TTGGTGTT~AAA~CACAJU~CATTTAT~ ~GT~GATTTTTCGCTTCAAA~TACAGTCCCC~TCTCGAAGCGACAAATTTT~TGA~AGATTACTGT~TTTCAAATTCC-GGAATT~CA6CA ATTTTCCATATTA~T~AC6TCT~AAATGA6AGG~CTGAT~CC~CATGTCGATTTA~6GGGCA~TAT~CATCATCTCAAAC~TCCATTT~TG~ TTT~C~AAATATGT~T~C~CCC6TA~TCGAC~TTCGT~ CGAGACTCAT6TGGC~AAAAC CTCATATATATCC6TCTA6AT~TAG TTTTTCT~TCGAT~TATA~AATTTAT~T~TTTAG~GAAAACTA~AAAAATCGAT6~TTCCTA~A6T~AAA~ACA~T~TTTT~TAAAAA

1400 T~CC~TTTACAGATTTTG~GTAAAAAGT~T~A~CC~T~TTTGAA6TT~TCAT~TTAAAAAAAA~AAAATA~A~ I l l ~

E A "R K S A N D T" T L A "T V A S N L E" P 1 G "R 1 Q H R T R" R T L " 15~ GCA~GCCCGGAAATCAGCAAATGA~AC~CA~TGGCCACCGTCGCTTCA~TCTGGAGCC~CCGCATTCAGATGA~ACGAGACGTACGC~

R G H L A K I Y A H H" M A S "D S R N 1600 AG~GG~CATTTAGCCAAGGTAG~GCTTTCkGACTTTTTCTAGTCATA~ATCGATA~Tf~GCAGAT~ATGC~T6CACTGGGCATCA6~CAGTCGCA

L V S" A S Q "D G K L I V ~" D S Y "T T N k 1700 ATTT~TGTCAGCTTCACAAGAC~GA~GCTCATCGTTTGGGACTCGTACACCACAAATA~GTGAG~TTTTTTGACGATTTTC~G~T~T~TT

V H A I" P L R "S S ~ V H T C" A Y A " 18~GATAAAAAA~AAAAAATTTAAAGAAATT~TTTGCATTTTTTTTCC~GTTCA~C~TTCCC~G~ATCATCGT6~TGA~ACT~CCTA~CT

P S G S F V A ' C G G . 19(]~CCAT¢GGGATCGT~GTCGCTTGT~A~T~CTG~ATTACTATTGTATTCAAA~TTCC~A6AGA~ACT~G~ATCT6GGTTTCTATT6AGA

L" D N I "C S I Y L~AT~G~TCTCGTAAATCTACACG~CAA~TTCAAAT~TTT~AATA~GT~C~TTCT~ATTTTTCCA~TCTCGAC~cATTTGCTC~TTT

S L K" T R E "G N V R V S R" E L P "~ H T G Y L S" C C R "F L D D N Q I" 21~ ACTCGCTG~AA~A~GC~AG~GAAAC~TGCGGGTGTCTCGTGAGCTTCCAGGACATACTGGATACTTGTC~TGTTGTCGATTC~TTGATGAC~CAAAT

V T S ' S G D H T C ~ TGTCA~TCTTCCGGAGATAT6ACTTG6T~6TTTTTTTTTTGAAAAATTTCATTTTTTTTAATTAAAG~AAATCCTA~AAA~ATCATA~TTATC~

I ~ cTA~TTT¢TAcTTTTCATTTTTTTTTTTc~cGTAATTT~A~CATcAGT~TATA~ccA~A~cAcAcAAAA~TA~GAAGT~c~AA~AGA~TTc~AT GTTGAATGTTGAT~AGTTTGTTTTCAAACTGGGCTCTTTTTGTGCCGCTTGATGAAATTGAGGTGGAGAG~GCAAA6~CGTGTG~TAAAA~ACAG~ A~AGCACAAAAG~CGGCCCGCGAA~A~G~AAA~T~CGGAC~C~ACAAG~C~A~GT~AAA~GA~CTC~AGACGTG~AG~GTACT~ ~GAAAAG~AAAAA~CCAG~/UU~A~G~AAGAAAGA~CAGA~AAC~CCAAAA~A~C~G~A~C~CAAAA~CAGA~T~1FAAAG CCA~ACGCTA~TCCGA~GC-(~CGCCACGAAA~AGAAG~AA~TCAAA~C~AAA~G~A~T~AG~AAAA~

2 8 ~ A T ~ T A ~ A ~ A A A ~ C A A A T C ~ T ~ G C ~ T G T ~ C ~ A ~ T A T ~ T C G T G G ~ A ~ T T T T ~ A ~ C A ~ C C T T T ~ C

A L ~ D I E T 6 Q ' Q C T 2900 CTCTACCATATCATATATATCACT~TTCGTA~TTATCCTA~TTCTCC~TTTTTTTTCCAGCGCTCTATGGGACATT~AAACC~TC~CAG~CACC

A F T G H T G" D V H "S L S l S P D" F R T "F I S G A C D A S A "K 3000 GCGTTCACCGGTCACACTGGTG~CGTCATGTCGCTTTCACTTTCTCCAG~CTTCCGCAC~TTCATCTCAGGAGCCTGTGACGCTTC~CG~TATA~

L ~ D I R D" G H C K Q T F P G H" E S D 31~ ATTTTTGAA~GTTGATTTTTTTTTAAATTTTTTAAATTT~AGTTGTGG~ATATCCGAGACGGCATGTGC~6CAAACG~CCCA~AcAcGAGTCAGAC

I N A V A F P S G N R F ' A T G S 3200 ATC~CGCTGTTGCCGTAAGTTTTATTATTTTG~GATT~TCAAAAA~AATC~TTTT~GCAGTTCTTCCCATC~GGAAA~C~TTCGCCACC~AT

D D A" T C R "L F D I R A D" Q E L "A H Y S H D N I I C G I T S V A F" 3~0 CCGACGATGCGA~ATGCCGATTGTTCGACATTCG~CTGATCA~CT~CCA~TATTCTCATGATAATATTATT~C~TCACTA~T6T~GCATT

S K S "G R l l F A G" Y D O "F N C N V ~ D" S H R Q E R A G ~W~CTCAAAATCGGGTCGCCTGTTGTTCGCC~ATACGACGACTTCAACTGC~TTTGGGATTCGA~CGACA~AG~AGCT~TG~CCTTT~CTC

V L A G H D N R V S C L 6" ~5~ CTTTTTTTG~U~ATTTTCCAA~TT~AAAG~ATGAJULAACT~TTGTTC~CC~TTTCAGGAGTATTG~CTGGTCACGAT~CC6AGTTTC~GTC~

V T E "O G H A V C T" G S ~ "D S F L K I ~" N • 3600 AGTCACCGA6GACGGTATGGCCGTG~CACA~ATCA~GGACTCGTTCCTC~GATC~G~GA/~ACATCAAATGTCTTCC~ATTCTCCA

ATTATTGATTG~TGAT~ATGACACTACGT~C~TTTTTC~C~AT~TCCC~AATTTTTTT~G~TCTTGAAACCCC~CTG~G~CTCA~CTTTC ~TCGCTATCCGCTATCCCG~CGCCCTCTTCCGCGTGCCCG6ATCTCTTTTTCTTCTCTCACCTCTTCTCATCGCTTTTTG~C~TACTTTCTA6TCTCT CTTC~TCGCCCTCCCCATATTACTTTCCTTGACGTTTGTTCGAATTTTCTCATC~TTTTTTTTACTATGT~AAAATTATTA~ATAGC~TTCTC~C TCTCTCTCTCTCATTTATTTTTATTTCACATT~CTGTGTCTCCCC~CCCCCCGCG~GCTTAATCTTTCTTCCTCTTTCACCACTCACTTTA~TCACAG ~ACATACACAT~CGCGCGCTCTCTTTCTCTCCCCGATCGTATTCTACT~CATGATATTCAT~CTACGACAAAAAATTTTATATATATGT~GACAAAT ~G~CTC~CGAG~ATATACA~TGATGGA~CCA~GC~CGC~A~TAC~TTTTGAAATCGAAAAG~6~CAC6CGCATCACT~TCCTAAAAC C~CCACATC~GAAAAGCAC~TA~AGATCCA~CT~TTATTATTTC~TC~CGATATAT~TTATTT~GCTTCTT11~TCTAT~CT~TATCA TCTTCTCTCA~TCTCGTC~CTACACT~TTT~T~ACTCCCCCCACAAATCTTCACCTCTTTT~GTCTTCTA~TATCCCAT~ATCC~TTTCC ~ACCGT~TC~C1~1]TCACACACACA/LAACCCCCTAA~TT~TTTTTCT~TTTCTTTT~CTCCTACCACCG~ATTT~T~G~C~CC~T ¢GCAAAA~AAAGATAAAAAAGTTCTCA~TATTCG6AT~CTGCCACTTCTT~CCTCTCTT~TCCCCCTC~CCCCCCACATC~T~TCCATCAC ~CTT6AAAAA~CATCCCCCATCCCC CCATCTCTTCTTTTC CC CAAAACACC CAAAAAATC~AT~CCCTTT~A~C~TTTTA~6TTTCAA T~CCAAA~C~ATAAAT~CT~CCTTAT~CCTC~GTGA~CTCAGTT~TCAGACAT~A~TCAAAATTA~ATCATTGTTCTCCT~C~ ~ C C T T G ~ G ~ C A ~ C G A ~ A T G A T G ~ C T C ~ C G T C G G C T ~ C T ~ T C ~ T A C C A ~ T C CGNTCCGCG~C~T~C6~TC

Figure 3. DNA sequence &nd predicted amino acid sequence of the C. ele~a~ p-subunit gene. The nuoleotides are numbered relative to the A of the ATG translat ional initiation codon. The p red ic~d amino acid sequence is shown above the nucleotide sequence. The stop codon is represented by an ~ r i s k . Conserved splice junctions are underlined.


M 1 i

• . : ?

• %

, [

• • , ~-

,2

. ~;

1 6 3 1 - -

2 2 1 - -

1 5 4 - -

7 5 - -

Figure 4. RNase protection analysis. A uniformly 3~P-labeled 1400 nucleotide antisense RNA transcript from the SmaI-BamHI fragment (Fig. 2{c), probe C) was hybridized with 50 ~g of total C. elegans RNA (lane I) or 50 ]~g of E~cherichia coli tRNA (lane 2), followed by diges- tion with RNase. In lane 3 the undigested probe was loaded. Size markers are shown on the left (in bases). Separate lanes from the same autoradiogram are shown.

m 9 - 5

m 7 . 5

~ ' ~ - , ' ~ - ~ 4 . 4

,,,~ .~.~ ~ N:

@ N Figure 5. Northern analysis of C. ele~ans poly(A) +

RNA. Each lane contained 1/~g of total C. elegans poly(A) + RNA. Separate strips from the same nitrocellulose filter were hybridized with different probes. Lane 1, probed with a random primed 0-3 kb BamHI- BglII fragment from fl~15Sl (Fig. 2(e), probe A). Lane 2, probed with a random primed 1 kb NarI-SacI fragment from fl).15S1 (Fig. 2(c), probe B). Lane 3, probed with a random primed 1 kb C. elega~s actin probe. DNA size markers are indicated on the right (in kb).

(d) Northern analysis

By probing a Nor thern blot of C. elegans poly(A) + RNA with the 358 base-pairs (bp) B a m H I - B f l I I fragment from fl215, which contains par ts of the last two coding exons of the fl-subunit gene, a strongly hybridizing band is observed (Fig. 5). The hybridizing messenger RNA has a length of approximate ly 2"8 kb and is relatively abundant when compared with actin mRNA (probes of similar specific act iv i ty were used). The same messenger RNA hybridizes to a 1 kb NarI-SacI fragment, derived from the 3' untranslated region (Fig. 4).

The coding sequence and the 5 ' -untranslated region (Fig. 2) add up to 1187 bp. Compared to the


I 2

2 1 - ~ , ~ . . ' ,:;~;

4 .3 ,' t~.'~

2"0 -- ~7 " j , ~ . .

1-4 - - ?£'~, :::; "

0 - 9 5 -- ' : : , .

0 . 5 6 - - ' '

3 4 5 6 7 B

Figure 6. Southern analysis of the C. elegans fl-subunit gene. A total of l0 ~g of C. elegans genomic DNA (lanes 3 to 8) or 0"2 pg offl215Sl DNA (lanes 1 and 2) was digested with excess SacI (lanes l, 3, 5 and 7) or HindIII (lanes 2, 4, 6, and 8). Subsequent gel electrophoresis and transfer to nitrocellulose filter was performed as described in Materials and Methods. Two different probes were used. Lanes l to 6 were probed with a random primed BamHI- BglII fragment from fl)d5S1 (Fig. 2(c), probe A) and hybridized under high (lanes 1 to 4) or low stringency conditions (lanes 5 and 6). Lanes 7 and 8 were hybridized with a random-primed bovine transducing cDNA probe using low stringency conditions. Separate strips are from the same nitrocellulose filter. DNA size markers are shown on the left (in kb).

length of the fl-mKNA (2'8 kb), as determined by Northern analysis, this leaves 1"6 kb of the mRNA unaccounted for. Not all the 5' and 3' non-coding sequences present in the mRNA have been identified. In the genomic sequence several potential splice donor and acceptor sites are present 5' and 3' of the coding region.

(e) Southern analysis

Using the 1"4 kb bovine transducin fl-subunit cDNA and the C. eleyans fl-gene 358 bp BamHI-BglII fragment as probe, Southern analysis of C. elegans DNA was performed.

A single band corresponding to the fl-subunit gene was detected in genomic Southern blots of C. elegans N2 DNA cut with SaeI and HindIII {Fig. 6). The same bands are detected at both high and low stringency with the C. elegans genomic probe and at low stringency with the bovine cDNA probe.

Since all genomic restriction fragments are accounted for by those detected in the C. elegans

genomic clone, it appears that the C. elegans genome contains no other sequence closely related to the G-protein fl-subunit gene.

(f) Chromosomal localization

The chromosomal localization of representative phage ,~ clones was analyzed by A. Coulson and J. Sulston (MRC, Cambridge, U.K.). Computer analysis of end-labeled restriction fragments of these clones was used to assign the fl-subunit gene to a previously mapped set of overlapping cosmids (contig: Coulson et al., 1986, 1988). The fl-subunit gene maps to the right arm of chromosome II, between sqt-1 and lin-29.

(g) Homology

Figure 7 compares the amino acid sequence encoded by the C. elegans fl-gene to the published amino acid sequences of human fll (Codina et al., 1986), human f12 (Fong et al., 1987) and Drosophila fl (Yarfitz et al., 1988). The C. elegans fl-protein shows 86% positional identity to ill, f12 as well as Drosophila ft. In this comparison, the amino- terminal 40 amino acids are the most divergent. Throughout the sequence, patches of divergence between the various fl-subunits are found (at position 174 to 180, 193 to 200, 213 to 219 and 298 to 303 in the amino acid sequence). The repetitive segmental structure described for the fl-subunit is maintained in C. elegans, as indicated in Figure 7. The patches of divergence are found predominantly at positions between these homologous segments. At the nucleic acid level, the similarity between the coding region of the C. elegans G-protein fl-subunit gene as compared to the coding regions of the human fll and f12, as well as the Drosophila fl-subunit gene, is 70% (data not shown).

4. Discussion

We describe here a G-protein fl-subunit gene from the nematode C. elegans. The deduced amino acid sequence of this protein is highly homologous (86~o) to fi-subunit sequences described from mammals (Codina et al., 1986; Fong et al., 1987) as well as from Drosophila (Yarfitz et al., 1988). The similarity of the C. elegans fl-gene coding sequence to the nucleotide sequence of other known fl-subunits is consider- ably lower (70~/o). The conservation of the amino acid sequence must be explained by strong similarity in function.

As pointed out by Fong et al. (1986), it is apparent that the fl-polypeptide consists of con- tiguous homologous segments, 43 to 47 amino acids in length, joined by non-homologous sequences of variable length. This repeated motif is found seven times in all fl-subunits described and might consti- tute a functional unit. Moreover, the same motif is found in the C-terminal half of two other, non- fi-subunit proteins: it is present six times in the


(1) (2) (3) (4)

MSELDQLRQEAEQLKSQIREARKSANDTTLATVASNLEPIGRIQMRTRRT -N---S ...... S--NA--D---A-C--S-LQA-TS

N---D---ACA-A--SQITN-ID-V-- E RN---D---ACG-S--TQITAG-D-V

(1) (2) (3) (4)

(1) (2) (3) (4)

(i) (2) (3) (4)

(1)

(2) (3) (4)

(1) (2) (3) (4)

(~) (2) (3) (4)

(i) (2) (3) (4)

:i~!iil

::::::::

i~-

-GN--- -GT--- -G~ ....

-A--T- -~-~S-

---N-Qi ---N--!

- - - s i

x,:.:

~ - ~ - - - D . . . . ~ N ~ - ~ : - ~ ~;~;~:~:~:~:~: ~::~

;YTTNKVHA

~ IETGQQCTA .... L-V-S ~i~! ~T-T ~ : . . . . . . TVG

I RDGMCKQT

S--R--

L . . . . . . L

SMRQERAGV T-KA--S-I

Consensus : ~,-GH-n-,,nnn- ( ..... ) --n~,c, nnn-Dn-n-aWD~,

Figure 7. Amino acid sequence comparison of fl-subunits from different species. C. elegans fl (1), Drosophila fl (2), human fll (3) and human f12 (4) are aligned according to the repetitive segmental pattern. Sequences are given in standard one-letter code. Amino acids included in the repeated structure are shaded. At the bottom of the aligned amino acid sequences, the consensus sequence for the repeated motif is depicted (a, hydrophobic amino acid; n, non-charged amino acid).

product of the yeast cell cycle gene CDCA (Yochem & Byers, 1987) and four times in the Drosophila Enhancer of Split (Hartley et al., 1988). Since the precise functions of the G-protein fl-subunit, the Enhancer of Split gene product and the CDC4 protein are still unclear, it is not known whether the presence of the repeated structure in these otherwise seemingly unrelated proteins reflects functional similarities. I t has been suggested that this structure of tandemly arranged homologous segments has evolved by duplication and divergence from a basic amino acid sequence (Fong et al., 1986). I f the r-gpeated segment indeed represents a functional entity, it is noteworthy that in the C. elegan8

fl-subunit gene introns are scattered throughout the repeated motifs. In the mouse fll gene, a similar phenomenon is observed. Moreover, the introns identified to date in the mouse map at sites different from the C. elegans introns (M. Simon, personal communication). In yeast (Whiteway et al., 1989) and Drosophila, the fl gene does not contain any introns in the coding sequence.

The results presented here suggest that there is only a single fl-subunit gene in C. elegans. From this gene, a single messenger RNA is transcribed. In Drosophila also only a single fl-gene could be detected, but evidence for differential splicing, not affecting the coding region, was found. Although the


possibility must be considered tha t C. elegans and Drosophila have additional, less homologous, fl-subunits, the evidence until now points to the presence of only one ~-subunit in these organisms. In mammals, there is evidence for at least four distinct fl-subunits (M. Simon, personal communication). Human and bovine fll have identical amino acid sequences. The same holds true for human and bovine f12. The presence of fll and f12 in all mammalian cell types tested, and the strong interspecies conservation supports the hypothesis tha t fll and f12 have distinct but related functions in trans- membrane signalling. In this regard, it is interesting tha t insects and nematodes seem to have only a single type of fl-subunit (as closely related to fll as to f12), while they apparent ly utilize t ransmembrane signalling pathways similar to those found in mammals.

From pertussis and cholera toxin labeling experiments, we have evidence for the existence of several a-subunits in C. elegans (data not shown). Similar findings have been reported for Drosophila (Yarfitz et al., 1988).

I f there is indeed a single type of fl-subunit inter- acting with different a-subunits in nematodes and Drosophila, the fl gene is likely to be an essential gene, implying tha t mutat ions in this gene may be lethal. Efforts to rescue lethals tha t map in the vicinity of the fl-subunit gene on C. elegans chromosome II may allow the identification of mutants with impaired fl-subunit gene expression. Their characterization will lead to further insight into G-protein action and the role of the fl-subunit in t ransmembrane signalling.

We thank John Sulston and Alan Coulson for the chromosomal assignment of the fl-subunit gene, Melvin Simon for communication of results prior to publication and Wouter Moolenaar and Anne Mahon for critical reading of the manuscript. This research was supported by the Netherlands Cancer Foundation (KWF) and the Netherlands Organization for the Advancement of Pure Scientific Research (NW0).

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Edited by S. Brenner

Note added in proof. These sequence data wilt appear in the EMBL/Genbank/DDBJ Nucleotide sequence Databases under the accession number X17497.

characterization of a g-protein β-subunit gene from the nematode caenorhabditis elegans

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