Cloning and expression of cDNA encoding human placental estrogen sulfotransferase

Using two oligoprimers derived from the bovine placental estrogen sulfotransferase sequence, we amplified a probe for human placental estrogen sulfotransferase. Using this probe to screen a human placental cDNA library constructed in λgt11, we isolated a cDNA clone of 1.3 kb encoding human estrogen sulfotransferase. DNA analysis predicts a protein of 295 amino acids with a calculated molecular weight of 34199. Alignment of the amino acid sequence with other sulfotransferases indicates that human placental estrogen sulfotransferase shares 68.6, 68.2 and 65.9% similarity with bovine placental, guinea pig adrenocortical, and rat liver estrogen sulfotransferase, respectively. It shows also 95.6, 57.6, 85.3, and 54.2% similarity to human phenol, human DHEA, rat phenol, and rat hydroxysteroid sulfotransferase, respectively. Transfection of expression vectors encoding human estrogen sulfotransferase and dehydroepiandrosterone (DHEA) sulfotransferase in human adrenal adenocarcinoma SW-13 cells indicates that estrogen sulfotransferase transforms estrone more specifically, whereas DHEA sulfotransferase is more specific for DHEA and pregnenolone.     

Cloning of cDNA encoding steroid 11 beta-hydroxylase (P450c11).

We have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11 beta-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage lambda vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11 beta-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.     

A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity.

Factor VIII (antihemophilic factor) is a high molecular weight plasma glycoprotein that participates in the blood clotting cascade. The recent cloning and sequence analysis of the cDNA encoding human factor VIII revealed an obvious domain structure for the protein, which can be represented as A1-A2-B-A3-C1-C2. We now report the DNA sequence analysis of porcine exons encoding the entire B domain and part of the A2 and A3 domains. We found an unusually high degree of porcine-human amino acid sequence divergence in the B region compared with the limited sequence available for other regions of the porcine factor VIII molecule. In addition to sequence divergence, there are numerous gaps in the porcine B domain totalling over 200 amino acids. Recombinant DNA techniques were used to effect the removal of large segments of DNA encoding the B domain from the full-length human factor VIII cDNA. These constructs directed the synthesis of biologically active factor VIII when introduced into mammalian cells despite the deletion of up to 38% of the factor VIII molecule.     

Deduced primary structure of the alpha subunit of the GTP-binding stimulatory protein of adenylate cyclase.

A bovine adrenal cDNA clone encoding the entire alpha subunit of the GTP-binding regulatory protein that stimulates adenylate cyclase (Gs) was isolated and sequenced. This cDNA directed the synthesis of the larger, 52-kDa form of the polypeptide in COS cells, even though the clone appeared to encode a 46-kDa protein. Comparison of the deduced amino acid sequence of Gs alpha with the alpha subunit of another G protein, transducin, revealed striking homologies.     

Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line

We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3'-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5' region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.     

Molecular cloning of the flavin-containing monooxygenase (form II) cDNA from adult human liver.

Complementary DNA (cDNA) clones encoding the adult human liver flavin-containing monooxygenase (FMO; dimethylaniline N-oxidase, EC 1.14.13.8) were isolated from lambda gt10 and lambda gt11 libraries. The cDNA libraries were screened with three synthetic 36-mer oligonucleotide probes derived from the nucleic acid sequence of the pig liver FMO cDNA. The deduced amino acid sequence for the adult human liver FMO was quite distinct from the pig liver FMO, and adult human liver FMO was designated form II (HLFMO II). The full-length cDNA sequence of HLFMO II [2119 base pairs (bp)] had an open reading frame of 1599 nucleotides, which encoded a 533-amino acid protein of Mr 59,179, a 5'-noncoding region of 136 nucleotides and a 3'-noncoding region of 369 nucleotides excluding the poly(A) tail. The deduced amino acid sequence of HLFMO II had 80% similarity with the rabbit liver FMO II but only a 52%, 55%, and 53% amino acid similarity with the rabbit liver (form I), the pig liver (form I), and fetal human liver (form I) FMOs, respectively. RNA analysis of adult human liver RNA showed that there was one HLFMO II mRNA species. Analysis of genomic DNA indicated that HLFMO II was the product of a single gene. These results indicated that the deduced amino acid sequence for HLFMO II contained highly conserved residues and suggested that FMO enzymes were closely related and, undoubtedly, derived from the same ancestral gene.     

Cloning of the cDNA for murine von Willebrand factor and identification of orthologous genes reveals the extent of conservation among diverse species

Interaction of von Willebrand factor (VWF) with circulating platelets promotes hemostasis when a blood vessel is injured. The A1 domain of VWF is responsible for the initial interaction with platelets and is well conserved among species. Knowledge of the cDNA and genomic DNA sequences for human VWF allowed us to predict the cDNA sequence for murine VWF in silico and amplify its entire coding region by RT-PCR. The murine VWF cDNA has an open reading frame of 8,442 bp, encoding a protein of 2,813 amino acid residues with 83% identity to human pre-pro-VWF. The same strategy was used to predict in silico the cDNA sequence for the ortholog of VWF in a further six species. Many of these predictions diverged substantially from the putative Reference Sequences derived by ab initio methods. Our predicted sequences indicated that the VWF gene has a conserved structure of 52 exons in all seven mammalian species examined, as well as in the chicken. There is a minor structural variation in the pufferfish Takifugu rubripes insofar as the VWF gene in this species has 53 exons. Comparison of the translated amino acid sequences also revealed a high degree of conservation. In particular, the cysteine residues are conserved precisely throughout both the pro-peptide and the mature VWF sequence in all species, with a minor exception in the pufferfish VWF ortholog where two adjacent cysteine residues are omitted. The marked conservation of cysteine residues emphasizes the importance of the intricate pattern of disulfide bonds in governing the structure of pro-VWF and regulating the function of the mature VWF protein. It should also be emphasized that many of the conserved features of the VWF gene and protein were obscured when the comparison among species was based on the putative Reference Sequences instead of our predicted cDNA sequences.     

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonucleotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cDNA libraries. A recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and DNA sequence analysis confirmed that this clone encodes human cathepsin B. The clone, designated pCB-1, has sequences for 81% of the coding region (for amino acid residues 50-252) together with approximately equal to 880 nucleotides of the 3' untranslated region of the mRNA. The DNA sequence also shows that the predicted carboxyl terminus of the coding sequence is longer than the mature protein by 6 amino acid residues. Southern blot analysis of restriction enzyme digests of human placental DNA revealed a simple pattern of hybridizing fragments using the cathepsin B coding sequence as probe. The result suggests that there is a single copy of cathepsin B gene per haploid genome.     

Characterization of the gene for the a subunit of human factor XIII (plasma transglutaminase), a blood coagulation factor.

Factor XIII (plasma transglutaminase, fibrin stabilizing factor) is a glycoprotein that circulates in blood as a tetramer (a2b2) consisting of two a and two b subunits. The primary structures of the a and b subunits of human factor XIII have been reported by a combination of cDNA cloning and amino acid sequence analysis. To establish the gene structure of the a subunit for factor XIII, several human genomic libraries were screened by using the cDNA encoding the a subunit as a probe. Among approximately equal to 5 x 10(7) recombinant phage, 121 have been shown to contain an insert encoding a portion of the a subunit. Twenty-five unique clones were then characterized by restriction mapping, Southern blotting, and DNA sequencing. Overlapping clones encoding the a subunit of factor XIII span greater than 160 kilobases. The gene was found to contain 15 exons separated by 14 introns. All the sequences of the introns at the intron-exon boundaries were GT-AG, which are the same as those found in other eukaryotic genes. DNA sequence analysis revealed that the activation peptide released by thrombin, the active site cysteine region, the two putative calcium-binding regions, and the thrombin cleavage site leading to inactivation are encoded by separate exons. This suggests that the introns may separate the a subunit into functional and structural domains. A comparison of the amino acid sequence deduced from the genomic DNA sequence with those deduced from cDNA or determined by amino acid sequence analysis of the plasma and placental proteins revealed apparent amino acid polymorphisms in six positions of the polypeptide chain of the a subunit.     

Cloning and sequence analysis of cDNA for human argininosuccinate lyase.

Using antibodies specific for argininosuccinate lyase (EC 4.3.2.1), we isolated two cDNA clones by screening a human liver cDNA library constructed in the lambda gt11 expression vector. The identity of these isolates was confirmed by in vitro translation of plasmid-selected mRNA. One of these isolates was used to rescreen the cDNA library and a 1565-base-pair (bp) clone was identified. The entire nucleotide sequence of this clone was determined. An open reading frame was identified which encoded a protein of 463 amino acids with a predicted molecular weight of 51,663. The clone included 115 bp of 5' untranslated sequence and 46 bp of 3' untranslated sequence. A canonical poly(A) addition site was present in the 3' end, 16 bp from the beginning of the poly(A) tract. Comparison of the deduced amino acid sequence of the human enzyme with that of the yeast enzyme revealed a 56% homology, when conservative amino acid changes were taken into consideration. The yeast protein is also 463 amino acids long, with a molecular weight of 51,944. By use of a genomic DNA panel from human-Chinese hamster somatic cell hybrids, the human gene was mapped to chromosome 7. Another hybridizing region, corresponding to a portion of the 5' end of the cDNA, was found on chromosome 22.     

Molecular cloning and sequence determination of rat preproenkephalin cDNA: sensitive probe for studying transcriptional changes in rat tissues.

A cDNA probe was prepared to investigate the regulation of proenkephalin biosynthesis in the rat. This was necessary because human and bovine proenkephalin cDNA were not sensitive enough for the accurate detection of preproenkephalin mRNA in tissues that contain low copy numbers of this message, such as the adrenal gland. The rat probe was prepared in the following manner. Preproenkephalin mRNA was enriched by sucrose gradient centrifugation of poly(A)-containing mRNA from rat brain and was used as a template for double-stranded cDNA synthesis. The resulting cDNA was inserted into the plasmid pBR322, and recombinant plasmids were used to transform Escherichia coli RR1 cells. A synthetic oligodeoxyribonucleotide (30 bases long) with a sequence that had previously been shown to be identical in bovine and human preproenkephalin cDNA was prepared to screen the clone bank. The plasmid with the longest cDNA insert (about 1200 bases) from the positive clones was isolated, and the sequence of the entire protein coding region was determined. Like the bovine and human gene products, rat preproenkephalin contains four [Met]enkephalin sequences and one copy each of [Leu]enkephalin, [Met]enkephalin-Arg6-Gly7-Leu8, and [Met]enkephalin-Arg6-Phe7. Rat preproenkephalin is 80% and 83% homologous to the bovine and human forms, respectively, at the nucleotide level and is 82% homologous to both species at the amino acid level. Rat preproenkephalin contains 269 amino acid residues, making it larger than the human (267 residues) and bovine (263 residues) precursors. The sensitivity for detection of rat preproenkephalin mRNA with the rat cDNA was several times greater than with the corresponding cDNAs from bovine and human sources.     

Study of cholesterol side-chain cleavage (20,22 desmolase) deficiency causing congenital lipoid adrenal hyperplasia using bovine-sequence P450scc oligodeoxyribonucleotide probes

Conversion of cholesterol to pregnenolone is mediated by the cholesterol side-chain cleavage (SCC) enzyme, P450scc. Deficient SCC activity causes congenital lipoid adrenal hyperplasia (also known as 20,22 desmolase deficiency), a potentially lethal defect in the synthesis of all steroid hormones. To probe for possible genetic defects causing this disease we synthesized four oligodeoxyribonucleotides containing 63 to 72 bases corresponding to portions of the bovine complementary DNA (cDNA) sequence for P450scc. The bovine oligonucleotides were labeled and used directly to probe Southern blots of normal human genomic DNA, revealing a pattern indicating there is a single P450scc gene in the human genome. Hybridization to Northern blots of normal human and bovine adrenal messenger RNA indicates that P450scc messenger RNA is about 2.0 kilobases long in both species. Hybridizations of the oligonucleotides to genomic DNA from three unrelated patients with SCC deficiency did not detect a deletion in the human P450scc gene. The bovine sequence oligonucleotides were then used to isolate a human P450scc cDNA clone. The isolated P450scc cDNA fragment contains 818 bases encoding 239 amino acids of the protein, the translation termination signal, and 98 bases of the 3' untranslated region. The sequence of this carboxy-terminal half of the human P450scc protein is 72% homologous with the bovine sequence and contains an additional amino acid not found in bovine P450scc; the human and bovine nucleotide sequences are 81% homologous. Repetition of the genomic DNA blotting studies with the cDNA probe gave the same results obtained with the bovine-sequence oligonucleotide probes, confirming that SCC deficiency is not due to a deletion in the regions of the P450scc hybridizing with the probes. Long, chemically synthesized heterologous sequence oligonucleotides containing unknown numbers of base mismatches with human sequences may thus be used to study human genes so that access to a cDNA is not necessary for such studies.     

Human pancreatic Beta-cell glucokinase: cDNA sequence and localization of the polymorphic gene to chromosome 7, band p 13

Summary The glucose phosphorylating enzyme glucokinase plays an important role in the regulation of glucose homeostasis. Studies in rodents indicate that pancreatic Beta cells and hepatocytes express different isoforms of this protein as a consequence of the presence of tissue-specific promoters and exon 1 sequences which are spliced to a shared group of nine exons which encode most of the mRNA and protein. Here, we report the isolation and characterization of cDNA clones encoding human Beta-cell glucokinase. The sequence of human Beta-cell glucokinase shows 97% amino acid identity with that of the cognate rat protein. We also mapped the human glucokinase gene to the short arm of chromosome 7 by analysing its segregation in a panel of reduced human mouse somatic cell hybrids. In situ hybridization to metaphase chromosomes confirmed the localization of the human glucokinase gene to chromosome 7 and indicated that it was in band p 13. A microsatellite DNA polymorphism that can be typed using the polymerase chain reaction was identified upstream of exon 1 a, the Beta-cell specific first exon. The glucokinase cDNA clone and highly informative DNA polymorphism will be useful for examining the role of this gene in the pathogenesis of diabetes mellitus.     

Isolation and sequence of a human apolipoprotein CII cDNA clone and its use to isolate and map to human chromosome 19 the gene for apolipoprotein CII.

cDNA clones encoding human apolipoprotein CII (apo CII) were identified by screening an adult human liver cDNA library with a mixed oligonucleotide probe corresponding to all possible codons for apo CII amino acid 6-10. One clone with an approximately equal to 500-base-pair (bp) insert, designated pCII -711, was selected for DNA sequence analysis. This clone contained a DNA sequence that corresponded with the previously reported amino acid sequence of apo CII with only minor differences. The DNA sequence specified a polypeptide of 79 amino acids, compared to the 78 amino acids previously reported. The pCII -711 clone contains a 36-bp DNA sequence upstream from that specifying the NH2-terminal threonine which, when read in frame, specifies the amino acid sequence Leu-Val-Leu-Leu-Val-Leu-Gly-Phe-Glu-Val-Gln-Gly and may be part of an apo CII signal peptide. The pCII -711 clone also contains a 144-bp region that corresponds to the 3' untranslated region of apo CII mRNA as well as a portion of the poly(A) tail. Clone pCII -711 was used to isolate and characterize by restriction endonuclease digestion the gene for apo CII from a human genomic library. In addition, through Southern blot analysis of DNA from human-rodent somatic cell hybrids, clone pCII -711 also was used to provisionally map the gene for apo CII to human chromosome 19.     

cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase.

We have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase (CPTase; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21), an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH2-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH2-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hamster somatic cell hybrids.     

Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation.

Protein S is a cofactor of activated protein C; together they function as a regulator of blood coagulation. A human liver cDNA library constructed in bacteriophage lambda gt11 was screened with DNA fragments from a full-length bovine cDNA clone encoding protein S. Several cDNA clones were isolated and sequenced. The combined cDNA sequences encoded the mature protein and 15 residues of the leader sequence when compared to bovine protein S. Human protein S is a single-chain protein consisting of 635 amino acids with 82% homology to bovine protein S. After an NH2-terminal gamma-carboxyglutamic acid-containing region, there is a short region with thrombin-sensitive bond(s), followed by a region with four repeat sequences that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the COOH-terminal portion of human protein S does not show any resemblance to serine proteases.     

Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat.

Cloned cDNAs encoding the precursor protein for gonadotropin-releasing hormone (Gn-RH) and prolactin release-inhibiting factor (PIF) were isolated from libraries derived from human and rat hypothalamic mRNA. Nucleotide sequence analyses predict precursor proteins of 92 amino acids for both species and show identity between the human placental and human hypothalamic precursor proteins. Whereas the Gn-RH peptide structure is completely conserved in human and rat, the PIF domain of the precursor displays 70% interspecies homology. Genomic analyses revealed the presence of a single Gn-RH-PIF gene in human and rat containing sequences corresponding to the cDNA distributed across four exons.     

Isolation and characterization of a cDNA clone for human ferritin heavy chain.

Ferritin, the main iron-storage protein, is composed of two partially homologous subunits, heavy (H) and light (L), with MrS of 21,000 and 19,000, respectively. We have isolated a cDNA clone for human ferritin H chains by screening a human lymphocyte cDNA library with synthetic oligodeoxyribonucleotides. The oligonucleotide sequences were derived from two pentapeptides found in human spleen ferritin. The selected clone hybridized to both probes and selected H-chain mRNA, but not L-chain mRNA, when hybridized to HeLa cell mRNA. These results indicate that the cloned DNA codes for a H chain of human ferritin. Since the amino acid sequence derived from the cloned DNA was almost identical to the partial amino acid sequence of a minor component found in human spleen ferritin, we conclude that the minor sequence found in human spleen ferritin must be a H subunit. Genomic analysis gives a complex pattern that suggests that ferritin H chains are encoded by a multigene family or have an unusually large number of exons.     

Two distinct genes for ADP/ATP translocase are expressed at the mRNA level in adult human liver.

Several clones hybridizing with a bovine ADP/ATP translocase cDNA were isolated from an adult human liver cDNA library in the vector pEX1. DNA sequence analysis revealed that these clones encode two distinct forms of translocase. In particular, two clones specifying the COOH-end-proximal five-sixths of the protein exhibit a 9% amino acid sequence divergence and totally dissimilar 3' untranslated regions. One of these cDNAs is nearly identical in sequence to an ADP/ATP translocase clone (hp2F1) recently isolated from a human fibroblast cDNA library [Battini, R., Ferrari, S., Kaczmarek, L., Calabretta, B., Chen, S. & Baserga, R. (1987) J. Biol. Chem. 262, 4355-4359], with three amino acid changes and a few differences in the 3' untranslated region. Another clone isolated from the pEX1 library contains a reading frame encoding the remaining, NH2-end-proximal, 37 amino acids of the translocase. This sequence differs significantly (14% amino acid sequence divergence) from the corresponding segment of hp2F1, and the 5' untranslated regions of the two clones are totally dissimilar. RNA transfer hybridization experiments utilizing the clones isolated from the pEX1 library revealed the presence in HeLa cells of three distinct mRNA species. The pattern of hybridization and the sizes of these mRNAs suggest a greater complexity of organization and expression of the ADP/ATP translocase genes in human cells than indicated by the analysis of the cDNA clones.     

Molecular cloning and characterization of cDNA encoding the GTP-binding protein alpha i and identification of a related protein, alpha h.

We have cloned and characterized cDNA encoding alpha i, the GTP-binding subunit of Gi, a protein that mediates hormonal inhibition of adenylate cyclase and hormonal regulation of other membrane functions. We have also identified cDNA encoding a putative protein, which we have named alpha h, that is highly homologous to alpha i but different from other known GTP-binding proteins. Both cDNAs were isolated from a bovine pituitary library. The cDNA encoding alpha i was identified by finding that the amino acid sequence determined for two tryptic peptides from alpha i agreed exactly with amino acid sequences deduced from the cDNA. We also determined the amino acid sequence of peptides derived from alpha o, a related 39-kDa protein purified from bovine brain. These sequences are approximately 75% identical to the sequence determined for alpha i. Southern blot analysis of bovine genomic DNA, using as probes radiolabeled cDNAs for alpha i, alpha h, and the alpha subunit of a related protein, transducin, showed that each probe recognized different genomic DNA fragments. Our results suggest a further level of complexity in the organization of the G-protein gene family, with multiple G proteins of very similar structural properties likely to be identified as products of distinct genes.