SMART MODE:

NORMAL GENOMIC

• Ouyang KX, Liu MQ, Pian RQ, Liu SS, Chen XY
• Isolation and analysis of alpha-expansin genes in the tree Anthocephalus chinensis (Rubiaceae).
• Genet Mol Res. 2013; 12: 1061-73
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• Expansins are cell wall-associated proteins that induce wall extension and relax stress by disrupting noncovalent bonds between cellulose microfibrils and cross-linking glycan chains, thereby promoting wall creep. Anthocephalus chinensis is a very fast-growing economically important tree found mainly in South Asia. Sixteen cDNAs, designated AcEXPA1 to AcEXPA16 (GenBank accession Nos. FJ417847, JF922686-JF922700) with corresponding genomic DNA sequences (GenBank accession Nos. GQ228823, JF922701-JF922715), were isolated by amplifying conserved domain binding with genomic walking and RACE techniques from four differential growth tissues in A. chinensis. These alpha-expansin homologues were highly conserved in size and sequence; they had the same sequence structures as an N-terminal signal peptide, three exons and two introns. Their amino acid alignment showed that A. chinensis expansin genes are divided into three subgroups: A, B and C. This study is the first report on expansin genes from A. chinensis. It will be used for a tissue-specific expression model and for studying the relationship between expansin genes, growth rate and wood quality of the xylem in this fast-growing tree.

• Landin MA, Shabestari M, Babaie E, Reseland JE, Osmundsen H
• Gene Expression Profiling during Murine Tooth Development.
• Front Genet. 2012; 3: 139-139
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• The aim of this study was to describe the expression of genes, including ameloblastin (Ambn), amelogenin X chromosome (Amelx), and enamelin (Enam) during early (pre-secretory) tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24 h intervals, starting at the 11th embryonic day (E11.5), and up to the 7th day after birth (P7). The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx, and Enam). Microarray results where validated using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), and translated proteins identified by Western-blotting. In situ localization of the Ambn, Amelx, and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially expressed (DE; p

• Dalla Valle L, Benato F, Rossi C, Alibardi L, Tschachler E, Eckhart L
• Deleterious mutations of a claw keratin in multiple taxa of reptiles.
• J Mol Evol. 2011; 72: 265-73
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• We have recently shown that homologs of mammalian hair keratins are expressed in the claws of the green anole lizard, Anolis carolinensis. To test whether reptilian hair keratin homologs are functionally associated with claws, we investigated the conservation of the prototypical reptilian hair keratin homolog, hard acidic keratin 1 (HA1), in representative species from all main clades of reptiles. A complete cDNA of HA1 was cloned from the claw-forming epidermis of the lacertid lizard Podarcis sicula, and partial HA1 gene sequences could be amplified from genomic DNA of tuatara, lizards, gekkos, turtles, and crocodiles. In contrast, the HA1 gene of the limbless slow worm, Anguis fragilis, and of two species of turtles contained at least one deleterious mutation. Moreover, an HA1 gene was undetectable in the softshell turtle, snakes, and birds. Mapping the presence and absence of HA1 onto the phylogenetic tree of sauropsids suggested that the HA1 gene has been lost independently in several lineages of reptiles. The species distribution of HA1 is compatible with the hypothesis of a primary function of HA1 in claws but also shows that the formation of reptilian claws does not strictly depend on this keratin.

• Katsu Y, Braun EL, Guillette LJ Jr, Iguchi T
• From reptilian phylogenomics to reptilian genomes: analyses of c-Jun and DJ-1 proto-oncogenes.
• Cytogenet Genome Res. 2009; 127: 79-93
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• Genome projects have revolutionized our understanding of both molecular biology and evolution, but there has been a limited collection of genomic data from reptiles. This is surprising given the pivotal position of reptiles in vertebrate phylogeny and the potential utility of information from reptiles for understanding a number of biological phenomena, such as sex determination. Although there are many potential uses for genomic data, one important and useful approach is phylogenomics. Here we report cDNA sequences for the c-Jun(JUN) and DJ-1(PARK7) proto-oncogenes from 3 reptiles (the American alligator, Nile crocodile, and Florida red-belly turtle), show that both genes are expressed in the alligator, and integrate them into analyses of their homologs from other organisms. With these taxa it was possible to conduct analyses that include all major vertebrate lineages. Analyses of c-Jun revealed an unexpected but well-supported frog-turtle clade while analyses of DJ-1 revealed a topology largely congruent with expectation based upon other data. The conflict between the c-Jun topology and expectation appears to reflect the overlap between c-Jun and a CpG island in most taxa, including crocodilians. This CpG island is absent in the frog and turtle, and convergence in base composition appears to be at least partially responsible for the signal uniting these taxa. Noise reduction approaches can eliminate the unexpected frog-turtle clade, demonstrating that multiple signals are present in the c-Jun alignment. We used phylogenetic methods to visualize these signals; we suggest that examining both historical and non-historical signals will prove important for phylogenomic analyses.

• Iwata T et al.
• Processing of ameloblastin by MMP-20.
• J Dent Res. 2007; 86: 153-7
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• Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro(2), Gln(130), Gln(139), Arg(170), and Ala(222). This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr(223), as well as the 17-kDa (Val(1)-Arg(170)) and 15-kDa (Val(1)-Gln(130)) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.

• Shintani S, Kobata M, Kamakura N, Toyosawa S, Ooshima T
• Identification and characterization of matrix metalloproteinase-20 (MMP20; enamelysin) genes in reptile and amphibian.
• Gene. 2007; 392: 89-97
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• Matrix metalloproteinase-20 (MMP20; enamelysin) is important for proteolytic processing of extracellular matrix (ECM) proteins during the formation of enamel and plays a critical role in proteolytic processing of amelogenin (AMEL), the most abundant enamel ECM protein. MMP20 might have played a role in the emergence of teeth, because jawless vertebrates with primordial teeth on their external skeletons may have possessed the MMP20 gene, and MMP20 and enamel ECM proteins are thought to have evolved together in a special relationship over time. Thus, an understanding of the molecular evolution of the MMP20 gene is important for elucidating the evolution of enamel and it is necessary to identify the orthologs of the MMP20 gene in non-mammals, as it has been identified in mammals. In the present study, orthologs of the MMP20 genes from a reptile (caiman) and an amphibian (African clawed toad) were cloned and characterized. Comparisons of the orthologs revealed that the MMP20 proteins were highly conserved throughout the evolution of tetrapods. Further, the caiman, toad, and mammalian MMP20 shared several unique features specific for MMP20, but not for other matrix metalloproteinases. In addition, the toad MMP20 gene was transcribed only in the upper jaw, presumably in teeth. These results suggest that MMP20 in a common ancestor of tetrapods might have been recruited for the processing of AMEL and conserved over 350 million years of evolution.

• Shintani S, Kobata M, Toyosawa S, Ooshima T
• Expression of ameloblastin during enamel formation in a crocodile.
• J Exp Zool B Mol Dev Evol. 2006; 306: 126-33
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• Ameloblastin is an enamel-specific protein that plays critical roles in enamel formation, as well as adhesion between ameloblasts and the enamel matrix, as shown by analyses of ameloblastin-null mice. In the present study, we produced two distinct antibodies that recognize the N-terminus and C-terminus regions of caiman ameloblastin, in order to elucidate the fate of ameloblastin peptides during tooth development. An immunohistochemical study using the antibodies showed that caiman ameloblastin was a tooth-specific matrix protein that may initially be cleaved into two groups, N- and C-terminal peptides, as shown in mammals. The distribution of the N-terminal peptides was much different from that of the C-terminal peptides during enamel formation; however, it was similar to that of mammalian ameloblastin. Although ameloblastin is thought to have a relationship with the enamel prismatic structure in mammals, in the caiman, which has non-prismatic enamel, functional ameloblastin has no relationship with any enamel structure. Consequently, it is suggested that ameloblastin has kept its original functions during the evolutionary transition from reptiles to mammals and that it has been conserved in both lineages during more than 200 million years of evolution. Our results support the notion that ameloblastin acts as a factor for ameloblast adhesion to enamel matrix, because distribution of the C-terminal peptides was consistently restricted on the surface layers of enamel matrix specimens ranging from immature to nearly completely mature. The principal molecules that provide the adhesive function are presumably C-terminal peptides.

• Lagos D, Ruiz MF, Sanchez L, Komitopoulou K
• Isolation and characterization of the Bactrocera oleae genes orthologous to the sex determining Sex-lethal and doublesex genes of Drosophila melanogaster.
• Gene. 2005; 348: 111-21
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• Here we report the isolation and characterization of the olive fruit fly Bactrocera oleae genes orthologous to the Drosophila melanogaster sex-determining genes Sex-lethal (Sxl) and doublesex (dsx). Fragments of the Sxl and dsx orthologous were isolated with RT-PCR. Genomic and cDNA clones were then obtained by screening a genomic library and separate male and female cDNA adult libraries using the RT-PCR products as probes in both cases. B. oleae Sxl gene (BoSxl) expresses the same pattern of transcripts which encode for a single common polypeptide in both male and female flies. The gene shares a high degree of similarity in sequence and expression to its Ceratitis capitata orthologous and does not appear to play a key regulatory role in the sex-determining cascade. B. oleae dsx gene (Bodsx) expands in a chromosomal region of more than 50 kb, with 6 exons-5 introns, producing different sex-specific mRNAs, according to the Drosophila model. The cDNA sequences are almost identical to the gene orthologous of Bactrocera tryoni. Four repeat elements identical to the D. melanogaster TRA/TRA-2 binding sites have been found in the untranslated region of the female-specific exon 4, predicting a common regulatory splicing mechanism in all studied species of Diptera.

• Miller HC, Belov K, Daugherty CH
• Characterization of MHC class II genes from an ancient reptile lineage, Sphenodon (tuatara).
• Immunogenetics. 2005; 57: 883-91
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• The organization and evolution of major histocompatibility complex (MHC) genes vary considerably among vertebrate lineages. MHC genes have been well characterized in mammals, birds, amphibians and fish, but little is known about their organization in reptiles, despite the fact that reptiles occupy an important phylogenetic position for understanding the evolutionary history of both mammalian and avian MHC genes. Here we describe the characterization of the first MHC class II B cDNA sequences from a non-avian reptile, the tuatara (Sphenodon spp.). Three class II B sequences were isolated from a tuatara cDNA library, and four additional partial sequences were isolated by reverse transcriptase-polymerase chain reaction. Six of these sequences appear to belong to the same gene family, which we have named SppuDAB. The remaining sequence (named SppuDBB) shares only 43.9% amino acid similarity with SppuDAB and thus appears to represent a separate gene family. SppuDBB may be a non-classical locus as it does not contain all the conserved residues expected of a classical MHC class II gene. Southern blot analysis indicates that only a single copy of SppuDBB exists in tuatara, but that multiple loci related to SppuDAB are present. The SppuDAB sequences have the highest amino acid similarity (57.2-62.4%) with class II B sequences from the spectacled caiman, but only 26.4-48.7% similarity with sequences from other vertebrates. The tuatara sequences do not strongly group with other reptile sequences on a phylogenetic tree, reflecting the antiquity of the Sphenodon lineage and the lack of closely related sequences for comparison.

• Woods A, James C, Underhill TM, Beier F
• Identification of the putative collagen X gene from the pufferfish Fugu rubripes.
• Gene. 2004; 342: 77-83
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• The type X collagen gene is the classical marker of hypertrophic chondrocytes. Mutations in the human collagen X gene cause metaphyseal chondrodysplasia type Schmid (MCDS). In order to gain insight into the evolution of collagen X genes, we identified a putative collagen X gene from the pufferfish Fugu rubripes. We demonstrated expression of the putative Fugu collagen X gene by reverse transcription-polymerase chain reaction (RT-PCR) with primers spanning intron 1. The Fugu collagen X gene shares a common gene structure and high amino acid identity with mammalian and chicken collagen X genes. Interestingly, we have found that most of the residues mutated in human MCDS are highly conserved in the Fugu gene. The availability of the Fugu collagen X gene sequence will be of value in the identification of functionally important residues within the protein and for delineating regulatory elements that control collagen X gene expression in chondrocytes.

• Musante L, Bartsch O, Ropers HH, Kalscheuer VM
• cDNA cloning and characterization of the human THRAP2 gene which maps to chromosome 12q24, and its mouse ortholog Thrap2.
• Gene. 2004; 332: 119-27
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• Characterization of a balanced t(2;12)(q37;q24) translocation in a patient with suspicion of Noonan syndrome revealed that the chromosome 12 breakpoint lies in the vicinity of a novel human gene, thyroid hormone receptor-associated protein 2 (THRAP2). We therefore characterized this gene and its mouse counterpart in more detail. Human and mouse THRAP2/Thrap2 span a genomic region of about 310 and >170 kilobases (kb), and both contain 31 exons. Corresponding transcripts are approximately 9.5 kb long. Their open reading frames code for proteins of 2210 and 2203 amino acids, which are 93% identical. By northern blot analysis, human and mouse THRAP2/Thrap2 genes showed ubiquitous expression. Transcripts were most abundant in human skeletal muscle and in mouse heart. THRAP2 protein is 56% identical to human TRAP240, which belongs to the thyroid hormone receptor associated protein (TRAP) complex and is evolutionary conserved up to yeast. This complex is involved in transcriptional regulation and is believed to serve as adapting interface between regulatory proteins bound to specific DNA sequences and RNA polymerase II.

• Wang Z, Peters B, Klussmann S, Bender H, Herb A, Krieglstein K
• Gene structure and evolution of Tieg3, a new member of the Tieg family of proteins.
• Gene. 2004; 325: 25-34
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• TGF beta-inducible immediate early gene, Tieg, belongs to the superfamily of Sp1-like transcription factors containing three C(2)H(2)-zinc finger DNA binding motifs close to the C-terminus. So far, Tieg1 and Tieg2 have been identified in human and mouse. We identified Tieg3, a new member of the Tieg protein family by screening a mouse cDNA library. Tieg3 has almost all the known features of the Tieg protein family: it shares a highly conserved C(2)H(2) zinc finger DNA binding domain and is 96% identical to Tieg2 and 86% to Tieg1, respectively. In addition, the three repression domains at the N-terminus, R1, R2 and R3 are conserved in all the Tiegs. Similar to Tieg1 and Tieg2, Tieg3 mRNA is up-regulated in response to TGF beta 1 treatment and can perform the Sp1 sites mediated repression of transcription. A 4 kilobase (kb) long transcript of mouse Tieg3 can be detected using Northern-blot analysis. The gene of mouse Tieg3 contains four exons. Due to the amino acid sequence similarity, mouse Tieg2 is regarded as an orthologue of human Tieg2. However, the mouse Tieg3 gene is localized in a conserved segment on mouse chromosome 12 corresponding to human Tieg2 on chromosome 2 with the same gene order. An interesting explanation for this apparent contradiction might be a homologous recombination leading to loci exchange between the mouse Tieg3 and Tieg2.

• Hansson MC, Wittzell H, Persson K, von Schantz T
• Characterization of two distinct aryl hydrocarbon receptor (AhR2) genes in Atlantic salmon (Salmo salar) and evidence for multiple AhR2 gene lineages in salmonid fish.
• Gene. 2003; 303: 197-206
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• The aryl hydrocarbon receptor (AhR) mediates the toxicity of several environmental contaminants, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin, and other halogenated hydrocarbons in vertebrates. This receptor initiates the transcription of several biotransformation enzymes, which in turn are responsible for causing severe harm to biological tissue. Here we describe the isolation and complete characterization of the first two AhR genes from the teleost fish Atlantic salmon (Salmo salar). The predicted amino acid sequences contain regions characteristic of other vertebrate AhRs including basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains but show little similarity to other vertebrate AhRs across the C-terminal half. Furthermore, they do not contain distinct Q-rich domains as found in the mammalian AhR, which is in line with previously described fish AhR genes. The salmon cDNAs encode 1106 and 1107 putative residues, respectively, approximately 50 amino acids longer than previously characterized AhR genes. Phylogenetic analyses demonstrated that the two salmon AhR sequences cluster within the AhR subfamily of the bHLH-PAS family, in a clade containing fish AhR2 genes. Although the two AhR2 forms are 92% identical at the amino acid level, the distribution of sequence differences and the presence of both forms in 30 tested individuals suggest that they are not allelic but derived from separate loci. Interestingly, they are not orthologs of the rainbow trout (Oncorhynchus mykiss) AhR2 alpha and beta genes and the new salmon loci are therefore here designated AhR2 gamma and AhR2 delta. In line with this, PCR with DNA from rainbow trout revealed a new trout AhR locus that was more similar to the two salmon genes than to the trout AhR2 alpha and beta genes, suggesting that the rainbow trout possesses at least three distinct AhR2 genes. The presence of multiple AhR genes in these species is probably a consequence of the genome duplications that occurred in the early evolution of fish and later also specifically in the salmonid lineage. Reverse transcription-PCR analyses revealed that both AhR2 gamma and AhR2 delta are transcribed in the liver, spleen and muscles of adult salmon.

• Thakur JK et al.
• A POLYCOMB group gene of rice (Oryza sativa L. subspecies indica), OsiEZ1, codes for a nuclear-localized protein expressed preferentially in young seedlings and during reproductive development.
• Gene. 2003; 314: 1-13
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• The SET domains are conserved amino acid sequences present in chromosomal proteins that contribute to the epigenetic control of gene expression by altering regional organization of the chromatin structure. The SET domain proteins are divided into four subgroups as categorized by their Drosophila members; enhancer of zeste (E(Z)), trithorax (TRX), absent small or homeotic 1 (ASH1) and supressor of variegation (SU(VAR)3-9). Homologs of all four classes have been characterized in yeast, mammals and plants. We report here the isolation and characterization of rice (Oryza sativa L. subspecies indica) cDNA, OsiEZ1, as a monocot member of this family. The OsiEZ1 cDNA is 3133 bp long with an ORF of 2799 bp, and the predicted amino acid sequence (895 residues) corresponds to a protein of ca. 98 kDa. All the characteristic domains known to be conserved in E(Z) homologs (subgroup I) of SET domain containing proteins are present in OsiEZ1. In the rice genome, a 7499 bp long OsiEZ1 sequence is split into 17 exons interrupted by 16 introns. Southern analysis indicates that OsiEZ1 is represented as single copy in the rice genome. Expression studies revealed that the OsiEZ1 transcript level was highest in rice flowers, almost undetectable in developing seeds of 1-2 days post-fertilization but increased significantly in young seeds of 3-5 days post-fertilization. The OsiEZ1 transcript was barely detectable in mature zygotic embryos, but its levels were significantly higher in callus derived from rice scutellum, somatic embryos and young seedlings. The OsiEZ1/GUS recombinant protein was confined to the nucleus in living cells of particle-bombarded onion peels. The expression of OsiEZ1 complemented a set1Delta Saccharomyces cerevisiae mutant that is impaired in telomeric silencing. We suggest that the nuclear-localized OsiEZ1 has a role in regulating various aspects of plant development, and this control is most likely brought about by repressing the activity of downstream regulatory genes.

• Ikemoto T, Park MK
• Identification and characterization of the reptilian GnRH-II gene in the leopard gecko, Eublepharis macularius, and its evolutionary considerations.
• Gene. 2003; 316: 157-65
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• To elucidate the molecular phylogeny and evolution of a particular peptide, one must analyze not the limited primary amino acid sequences of the low molecular weight mature polypeptide, but rather the sequences of the corresponding precursors from various species. Of all the structural variants of gonadotropin-releasing hormone (GnRH), GnRH-II (chicken GnRH-II, or cGnRH-II) is remarkably conserved without any sequence substitutions among vertebrates, but its precursor sequences vary considerably. We have identified and characterized the full-length complementary DNA (cDNA) encoding the GnRH-II precursor and determined its genomic structure, consisting of four exons and three introns, in a reptilian species, the leopard gecko Eublepharis macularius. This is the first report about the GnRH-II precursor cDNA/gene from reptiles. The deduced leopard gecko prepro-GnRH-II polypeptide had the highest identities with the corresponding polypeptides of amphibians. The GnRH-II precursor mRNA was detected in more than half of the tissues and organs examined. This widespread expression is consistent with the previous findings in several species, though the roles of GnRH outside the hypothalamus-pituitary-gonadal axis remain largely unknown. Molecular phylogenetic analysis combined with sequence comparison showed that the leopard gecko is more similar to fishes and amphibians than to eutherian mammals with respect to the GnRH-II precursor sequence. These results strongly suggest that the divergence of the GnRH-II precursor sequences seen in eutherian mammals may have occurred along with amniote evolution.

• Vazza G, Picelli S, Bozzato A, Mostacciuolo ML
• Identification and characterization of C3orf6, a new conserved human gene mapping to chromosome 3q28.
• Gene. 2003; 314: 113-20
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• This study reports the characterization of a novel human gene, chromosome 3 open reading frame 6 (C3orf6), mapped to chromosome 3q28, within the critical region of hereditary spastic paraplegia SPG14 locus. Based on computational "spliced" EST alignment and RT-PCR, two C3orf6 transcript variants were identified. The longer C3orf6 transcript contains a 1449-nt ORF, encoding a protein of 482 aa, while the shorter variant contains a 921-nt ORF, encoding for a protein of 306 aa. C3orf6 gene is organised on 12 exons and the shorter transcript comes from an alternative splicing event skipping exon 6. The two mRNA are differentially expressed in brain and in several other human tissues with a predominant level for the shorter transcript. By database analysis, EST assembling and RT-PCR, we identified the transcripts of mouse and rat C3orf6 orthologous genes. The involvement of C3orf6 in the spastic paraplegia was investigated by sequencing all coding exons and flanking sequences in the SPG14 family, excluding the presence of causative mutations.

• Spassov DS, Jurecic R
• Mouse Pum1 and Pum2 genes, members of the Pumilio family of RNA-binding proteins, show differential expression in fetal and adult hematopoietic stem cells and progenitors.
• Blood Cells Mol Dis. 2003; 30: 55-69
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• Self-renewal is the common functional property of all types of stem cells and is thought to be regulated by unknown conserved intrinsic and extrinsic molecular mechanisms. Recently, an evolutionarily conserved Pumilio family of RNA-binding proteins that regulate asymmetric cell division was found to be essential for stem cell maintenance and self-renewal in Drosophila and Caenorhabditis elegans. Based on conserved function in invertebrates and lower vertebrates it was recently proposed that an ancestral function of Pumilio proteins is to support proliferation and self-renewal of stem cells. This raises an interesting possibility that Pumilio could be part of evolutionarily conserved intrinsic molecular mechanism that regulates self-renewal of mammalian stem cells. Here we describe cloning and comparative sequence analysis of Pum1 and Pum2 genes, mouse members of the Pumilio family, and for the first time demonstrate expression of Pumilio genes in mammalian hematopoietic stem cells (HSC). Pum1 and Pum2 share 51 and 55% overall similarity with the fly Pum, whereas their RNA-binding domains show a very high degree of evolutionary conservation (86-88% homology). Both genes are expressed in a variety of tissues suggesting that they have widespread function. During blood cell development Pum1 and Pum2 exhibit differential expression in cell populations enriched for HSC and progenitors. Both genes are highly transcribed in populations of adult HSC (Rho-123(low)Sca-1(+)c-kit(+)Lin(-) cells). In a more heterogeneous population of HSC (Lin(-)Sca-1(+)) and in progenitors (Lin(-)Sca-1(-) cells) Pum1 is not transcribed, whereas Pum2 expression is significantly down-regulated. Ongoing in vitro and in vivo functional analysis of mouse Pumilio genes will help to elucidate the biological role of mammalian Pumilio genes and determine whether they play any role in maintenance of mammalian stem cells, such as HSC.

• Benachenhou N, Massy I, Vacher J
• Characterization and expression analyses of the mouse Wiskott-Aldrich syndrome protein (WASP) family member Wave1/Scar.
• Gene. 2002; 290: 131-40
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• Characterization of multiprotein complexes involved in actin remodeling and cytoskeleton reorganization is essential to understand the basic mechanisms of cell motility and migration. To identify proteins implicated in these processes, we have isolated the mouse Wave1/Scar gene, a member of the Wiskott-Aldrich syndrome protein (WASP) family. The mouse Wave1 gene was physically localized on chromosome 10 and spans over 12 Kb comprising eight exons and seven introns. The mouse Wave1 complementary DNA encodes a predicted 559 amino acid protein, with a SCAR homology domain, a basic domain, a proline-rich region, a WASP homology domain and an acidic domain conserved in the orthologous proteins. The Wave1 transcription initiation site was mapped 210 base pairs upstream of the ATG translational start site. The presumptive proximal promoter contains putative consensus binding sites for E2 basic helix-loop-helix transcription factors, hepatocyte nuclear factor-3beta, S8 homeodomain protein, zinc finger transcription factor MZF-1, and an interferon-stimulated response element. Northern analysis demonstrated a strong expression of a unique approximately 2.6 Kb Wave1 transcript in brain tissue, and in situ hybridization showed restricted expression to Purkinje cells from the cerebellum and pyramidal cells from the hippocampus. Characterization and expression analyses of the murine Wave1 gene provide the basis toward functional studies in mouse models of the role of Wave1 in neuronal cytoskeleton organization.

• Carlone DL, Hart SR, Ladd PD, Skalnik DG
• Cloning and characterization of the gene encoding the mouse homologue of CpG binding protein.
• Gene. 2002; 295: 71-7
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• Human CpG binding protein (CGBP) is a ubiquitously-expressed transcriptional activator that binds specifically to unmethylated CpG motifs. Several protein domains have been identified within CGBP including two plant homeodomains (PHD), acidic and basic regions, a coiled-coil domain, as well as a CXXC DNA-binding domain. The global function of CGBP remains unclear, although failure to express CGBP results in embryonic lethality in mice. This study reports the identification and characterization of the murine CGBP gene locus. A 2509 bp murine CGBP cDNA was cloned and nucleotide sequence determined. Comparison of the mouse and human CGBP sequences revealed 86% identity at the nucleotide level and 96% identity at the amino acid level. Examination of the deduced translation product revealed that the PHD, CXXC, coiled-coil, and basic domains are identical between mouse and human, while the acidic region exhibits approximately 90% identity with its human counterpart. A single murine CGBP transcript of approximately 2.6 kb was detected in a wide variety of adult tissues as well as embryonic stem cells. Analysis of the mouse gene locus revealed a relatively small gene spanning approximately 5 kb and comprised of 15 exons. Examination of the human CGBP gene showed a similar size and structure with identical intronic splice sites. In contrast to the human CGBP gene, which is located 800 bp upstream of the MBD1 gene, analysis of the murine CGBP gene locus failed to detect the murine MBD1 gene within several kilobases of the CGBP coding region.

• Stohr H, Berger C, Frohlich S, Weber BH
• A novel gene encoding a putative transmembrane protein with two extracellular CUB domains and a low-density lipoprotein class A module: isolation of alternatively spliced isoforms in retina and brain.
• Gene. 2002; 286: 223-31
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• We report herein the cDNA cloning of a novel retina and brain specific gene from mouse and human encoding a putative transmembrane protein with an N-terminal signal sequence and two conserved extracellular CUB domains followed by a single copy of the low-density lipoprotein class A (LDLa) module. The mouse and human genes, termed NETO1 (neuropilin and tolloid like-1), display sequence identities of 87% at the nucleotide and 95% at the protein level. The human NETO1 gene comprises 13 exons on chromosome 18q22-q23 and gives rise to three different mRNA isoforms. Two alternative leader exons 1a and 1b generate transcripts that translate into putative signal peptides with individual sequence composition but otherwise do not affect the primary structure of the mature NETO1 protein. Usage of the internal exon 5 is restricted to the retinal tissue and generates a truncated transcript that codes for a putative soluble protein, termed sNETO1, with only one copy of the CUB domain while lacking the LDLa module. NETO1 exhibits 57% identity to the deduced amino acid sequence of a non-annotated nucleotide sequence in the GenBank database, therefore designated NETO2. Both NETO1 and NETO2 share an identical and unique domain structure thus representing a novel subfamily of CUB- and LDLa-containing proteins. The cytoplasmic domains of NETO1 and NETO2 are not homologous to other known protein sequences but contain a conserved FXNPXY-like motif, which is essential for the internalization of clathrin coated pits during endocytosis or alternatively, may be implicated in intracellular signaling pathways.

• Yuasa HJ, Nakatomi A, Suzuki T, Yazawa M
• Genomic structure of the sponge, Halichondria okadai calcyphosine gene.
• Gene. 2002; 298: 21-7
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• Calcyphosine is an EF-hand Ca(2+)-binding protein, which was first isolated from the canine thyroid. It is phosphorylated in a cyclic AMP (cAMP)-dependent manner; then it is thought to be implicated in the cross-signaling between the cAMP and calcium-phosphatidylinositol cascades. Here, we isolated the DNA complementary to RNA (cDNA) of an EF-hand Ca(2+)-binding protein from the sponge, Halichondria okadai and determined its genomic structure. The deduced sequence of the sponge Ca(2+)-binding protein showed significant similarity (about 40% identity) with those of mammal calcyphosines, and the intron positions were well conserved between the sponge and human calcyphosine genes. We considered that the isolated cDNA was that of sponge calcyphosine, and that sponge and mammalian calcyphosines evolved from a common ancestor gene. Recent cDNA projects have revealed that a calcyphosine cDNA is also expressed by human, mouse, and the ascidia. These cDNAs have more than 60% identity with sponge calcyphosine and each other, and all are composed of 208 amino acid residues. On the constructed phylogenetic trees, calcyphosines are essentially divided into two groups, types-I and -II calcyphosines. Type-I calcyphosine may be specific to mammals, and type-II is widely distributed among metazoan species. This suggests that type-II calcyphosine is a rather ancient gene with some essential function.

• Aronovich EL, Johnston JM, Wang P, Giger U, Whitley CB
• Molecular basis of mucopolysaccharidosis type IIIB in emu (Dromaius novaehollandiae): an avian model of Sanfilippo syndrome type B.
• Genomics. 2001; 74: 299-305
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• Sanfilippo syndrome type B, or mucopolysaccharidosis (MPS) IIIB, is an autosomal recessive disease caused by a deficiency of lysosomal alpha-N-acetylglucosaminidase (NAGLU). In Dromaius novaehollandiae (emu), a progressive neurologic disease was recently discovered, which was characterized by NAGLU deficiency and heparan sulfate accumulation. To define the molecular basis, the sequences of the normal emu NAGLU cDNA and gene were determined by PCR-based approaches using primers for highly conserved regions of evolutionarily distant NAGLU homologues. It was observed that the emu NAGLU gene is structurally similar to that of human and mouse, but the introns are considerably shorter. The cDNA had an open reading frame (ORF) of 2259 bp. The deduced amino acid sequence is estimated to share 64% identity with human, 63% with mouse, 41% with Drosophila, 39% with tobacco, and 35% with the Caenorhabditis elegans enzyme. Three normal and two affected emus were studied for nucleotide sequence covering the entire coding region and exon-intron boundaries. Unlike the human gene, emu NAGLU appeared to be highly polymorphic: 19 variations were found in the coding region alone. The two affected emus were found to be homozygous for a 2-bp deletion, 1098-1099delGG, in exon 6. The resulting frameshift predicts a longer ORF of 2370 bp encoding a polypeptide with 37 additional amino acids and 387 altered amino acids. The availability of mutation screening in emus now permits early detection of MPS IIIB in breeding stocks and is an important step in characterizing this unique, naturally occurring avian model for the development of gene transfer studies.

• Tsuchida S, Yamada R, Ikemoto S, Tagawa M
• Molecular cloning of a cDNA coding for feline liver xanthine dehydrogenase.
• J Vet Med Sci. 2001; 63: 353-5
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• A cDNA coding for feline liver xanthine dehydrogenase (XDH, EC 1.1.204) was amplified by RT-PCR and cloned for determining the sequence. The clones contained an open reading frame of 4002 base pairs encoding 1333 amino acid residues. The calculated molecular weight and isoelectric point were approximately 146 kDa and 7.0. Comparison of the deduced amino acid sequences indicated remarkable high homology, i.e., the amino acid residues of feline XDH shared approximately 90%, 87%, 87% and 86% identity with those of human, bovine, rat and mouse, respectively. The anino acid sequences of two putative iron-sulfur centers, one NAD binding site and one molybdenum binding site were well conserved among mammalian animals.

• Ma Y et al.
• ATP citrate lyase in the glaucocystophyte alga Cyanophora paradoxa is a cytosolic enzyme: characterisation of the gene for the large subunit at the cDNA and genomic levels.
• Mol Genet Genomics. 2001; 266: 231-8
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• The single-copy gene for the large subunit of ATP citrate lyase (ACL) from the alga Cyanophora paradoxa was characterized at the cDNA and genome levels. The gene product showed high sequence similarity to its mammalian counterparts, but is smaller in size, as is also found for the fungal subunit Acl1 and, most probably, for the corresponding subunit from Arabidopsis thaliana. The C. paradoxa gene is interrupted by at least 12 introns of 53-65 bp with conserved border and branchpoint sequences, and the product lacks a stroma-targeting peptide. Enzyme activity was found in the cytosol of C. paradoxa but not in the plastid (cyanelle) fraction. This is in contrast to the subcellular distribution of ATP citrate lyases in higher plants, where both chloroplast and cytosolic enzymes have been reported.

• Yamamoto A et al.
• Molecular cloning and expression of the cDNA encoding feline granulocyte colony-stimulating factor.
• Gene. 2001; 274: 263-9
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• Both genomic DNA and cDNA of the feline granulocyte colony-stimulating factor (G-CSF) gene were cloned from CRFK cells. Southern blot analysis showed that the haploid genome contains a single copy of the G-CSF gene. The RT-PCR analysis of several feline cell lines revealed expression of G-CSF mRNA in response to lipopolysaccharide stimulation. Sequence analysis of genomic and cDNA clones indicated that the intron-exon junction structure is conserved between the human and the feline G-CSF genes. The G-CSF coding region encodes a predicted protein of 195 amino acids including a signal sequence of 21 amino acids. The feline G-CSF amino acid sequence shares a high degree of identity with the canine (90.8%), human (87.4%), ovine (83.9%), bovine (82.8%), porcine (80.5%), murine (70.7%) and rat (66.8%) G-CSF. The feline G-CSF expressed in insect cells using recombinant baculovirus vector was biologically active as measured in a proliferation assay using NFS-60 cells and an induction assay of leukocytes in cats.

• Dewilde S, Van Hauwaert ML, Vinogradov S, Vierstraete A, Vanfleteren J, Moens L
• Protein and gene structure of a chlorocruorin chain of Eudistylia vancouverii.
• Biochem Biophys Res Commun. 2001; 281: 18-24
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• The polychaete annelid, Eudistylia vancouverii, contains as oxygen carrier a hexagonal bilayer (HBL) chlorocruorin. One of the globin chains, chain a1, has 142 amino acids (Mr 16,054.99) and its sequence deviates strongly from other nonvertebrate globin sequences. Unprecedented, it displays a Phe at the distal position E7 as well as at position B10, creating a very hydrophobic heme pocket probably responsible for the low oxygen affinity of the native molecule. Phylogenetic analysis of annelid globin chains clearly proves that globin chain a1 belongs to type I of globin chains having a pattern of 3 cysteine residues essential for the aggregation into a HBL structure. The gene coding for globin chain a1 is interrupted by 2 introns at the conserved positions B12.2 and G7.0. Based on protein and gene structure it can therefore be concluded that the globin chains of chlorocruorins are not fundamentally different from other annelid globin chains.

• Grailhe R, Grabtree GW, Hen R
• Human 5-HT(5) receptors: the 5-HT(5A) receptor is functional but the 5-HT(5B) receptor was lost during mammalian evolution.
• Eur J Pharmacol. 2001; 418: 157-67
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• We have isolated from a human genomic library the human 5-hydroxytryptamine 5-HT(5A) and 5-HT(5B) genes. The human 5-HT(5A) gene encodes a protein with similar characteristics to its mouse homologue. When expressed in monkey COS-7 cells, the human 5-HT(5A) receptor displayed a high affinity for tritiated 5-carbamidotryptamine ([3H]5-CT; K(D)=2.8 nM) and iodinated lysergic acid diethylamide ([125I]LSD; K(D)=187 pM). These binding sites displayed the following displacement profile: Ergotamine>Methiothepin>5-CT, Ritanserin>5-HT. Reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the presence of human 5-HT(5A) mRNA in the central nervous system but not in peripheral organs. When expressed in Xenopus oocytes, the 5-HT(5A) receptor was able to couple to the inwardly rectifying K(+) channel, GIRK(1). In contrast to the human 5-HT(5A) gene and the mouse 5-HT(5B) gene, the human 5-HT(5B) gene does not encode a functional protein because its coding sequence is interrupted by stop codons. Our results suggest, therefore, that the 5-HT(5B) receptor has been lost during evolution after the divergence between rodents and primates. The 5-HT(5B) receptor is the first example of a brain-specific protein that is absent in human.

• MacDougall M et al.
• Cloning, characterization and immunolocalization of human ameloblastin.
• Eur J Oral Sci. 2000; 108: 303-10
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• Amelogenesis imperfecta is a broad classification of hereditary enamel defects, exhibiting both genetic and clinical diversity. Most amelogenesis imperfecta cases are autosomal dominant disorders, yet only the local hypoplastic form has been mapped to human chromosome 4q between D4S242 1 and the albumin gene. An enamel protein cDNA, termed ameloblastin (also known as amelin and sheathlin), has been isolated from rat, mouse and pig. Its human homolog has been mapped to chromosome 4q21 between markers D4S409 and D4S400, flanking the local hypoplastic amelogenesis imperfecta critical region. Therefore, ameloblastin is a strong candidate gene for this form of amelogenesis imperfecta. To facilitate genetic studies related to this dental disease, we isolated and characterized a human ameloblastin cDNA. A human third molar cDNA library was screened and two ameloblastin clones identified. Nucleotide sequencing of these cDNAs indicated alternative splicing of the putative open reading frame, use of different polyadenylation signals, and a high degree of similarity to reported rat, mouse and porcine cDNAs. Immunohistochemistry studies on embryonic human teeth using an antibody to recombinant ameloblastin indicated ameloblastin expression by ameloblasts with localization in the enamel matrix associated with the sheath structures.

• Zuccolotto PD, Harrison GA, Deane EM
• Cloning of marsupial T cell receptor alpha and beta constant region cDNAs.
• Immunol Cell Biol. 2000; 78: 103-9
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• Partial cDNAs encoding the tammar wallaby (Macropus eugenii) T cell receptor alpha constant region (TCRalphaC) and T cell receptor beta constant region (TCRbetaC) were obtained using reverse transcriptase-coupled polymerase chain reaction (RT-PCR). These PCR products were used to screen a brushtail possum (Trichosurus vulpecula) lymph node cDNA library, resulting in the isolation of clones containing the complete coding regions for TCRalphaC and TCRbetaC. These constitute the first marsupial T cell receptor sequences to have been elucidated. Sequence analysis of the T. vulpecula constant region revealed a considerable level of sequence identity with TCR of other species, particularly eutherian mammals, at both the nucleic acid and amino acid levels. At the nucleotide level, 65.8% sequence identity was calculated for the T. vulpecula and human TCRalphaC sequences, with 55.9% identity at the amino acid level. For TCRbetaC, the T. vulpecula and human beta1 sequence identity at the nucleotide level was 75.1% and at the amino acid level, 67.0%. Phylogenetic analyses based on the T. vulpecula sequences indicated that these sequences are basal to, but also most closely related with, TCRalphaC and TCRbetaC homologues from eutherian mammals, consistent with the current views of both mammalian and TCR evolution.

• Byrne KA, Lehnert SA, Johnson SE, Moore SS
• Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus.
• Gene. 1999; 239: 317-24
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• Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1, 4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.

• Bedekar A, Zink RM, Sherman DH, Line TV, Van Pilsum JF
• The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution.
• Comp Biochem Physiol B Biochem Mol Biol. 1998; 119: 677-90
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• The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared. The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical. Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match. Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces. We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes. The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine + cytosine (G + C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.

• Rescan PY
• Identification of a fibroblast growth factor 6 (FGF6) gene in a non-mammalian vertebrate: continuous expression of FGF6 accompanies muscle fiber hyperplasia.
• Biochim Biophys Acta. 1998; 1443: 305-14
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• FGF6, a member of the fibroblast growth factor (FGF) family, is specifically expressed in developing skeletal muscle and may participate in muscle maintenance and regeneration. Until now, no convincing evidence for the existence of an FGF6 gene in non-mammalian vertebrates has been put forward. Only a hybrid growth factor containing features characteristic of both FGF4 and FGF6 has been identified in frogs and chickens, suggesting that the step of duplication which created FGF4 and FGF6 took place with the emergence of mammals. In this study, we report the isolation and characterization of a genomic clone encoding the trout (Oncorhynchus mykiss) fibroblast growth factor 6 (TFGF6). An initial cDNA clone was generated by PCR amplification using degenerate oligo primers corresponding to a conserved region of protein found in the mouse and human homologs. The screening of a genomic library with the cloned PCR product led to the isolation of a clone composed of three exons encoding a putative protein of 206 amino acids which exhibits a potential signal peptide and shows 64.6 and 63.6% similarity with mouse and human FGF6, respectively (77% over the carboxy two-thirds of the protein) and only 46.5% similarity with mouse and human FGF4 (62% over the carboxy two-thirds of the protein). The splice position of the three exons was found to be analogous to the human and mouse FGF6 and the start translation site of TFGF6 was preceded by a long stretch of nucleotides that is highly and specifically conserved in mammalian FGF6. Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the expression pattern of TFGF6 is close to that of mammals, TFGF6 transcripts being present in muscle (fast-twitch and to a lesser extent slow-twitch fibers), heart, testis and brain. Interestingly, the prolonged phase of muscle fiber hyperplasia which occurs in trout is accompanied by the lasting expression of TFGF6 up to the adult stage suggesting that TFGF6 may participate in the continuous generation of muscle fibers within the myotomal musculature of post larval animals.

• Girondot M, Delgado S, Laurin M
• Evolutionary analysis of "hagfish amelogenin".
• Anat Rec. 1998; 252: 608-11
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• Hagfishes lack mineralized tissues and teeth. Part of a cDNA strand, allegedly from amelogenin, the major gene involved in enamel formation in mammals, has recently been cloned in a hagfish (Slavkin and Diekwish, Anat. Rec., 1996;245:131-150). This cloning is of great interest because it could change the current view about the evolution of mineralized tissues, but no phylogenetic analysis of this piece of DNA has been made by the authors. Phylogenetic analysis of this part of cDNA has been conducted using both phenetic and cladistic methods. The cDNA amplified in hagfish does not fit with a nonmammalian origin but fits well with a degraded rodent sequence. The gene cloned in hagfish is probably of mammalian origin due to contamination during PCR.

• Mannen H, Tsoi SC, Krushkal JS, Li WH, Li SS
• The cDNA cloning and molecular evolution of reptile and pigeon lactate dehydrogenase isozymes.
• Mol Biol Evol. 1997; 14: 1081-7
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• The cDNAs encoding lactate dehydrogenase isozymes LDH-A (muscle) and LDH-B (heart) from alligator and turtle and LDH-A, LDH-B, and LDH-C (testis) from pigeon were cloned and sequenced. The evolutionary relationships among vertebrate LDH isozymes were analyzed. Contrary to the traditional belief that the turtle lineage branched off before the divergence between the lizard/alligator and bird lineages, the turtle lineage was found to be clustered with either the alligator lineage or the alligator-bird clade, while the lizard lineage was found to have branched off before the divergence between the alligator/turtle and bird lineages. The pigeon testicular LDH-C isozyme was evidently duplicated from LDH-B (heart), so it is not orthologous to the mammalian testicular LDH-C isozymes.

• Hu CC et al.
• Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins.
• J Dent Res. 1997; 76: 648-57
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• Sheath proteins designate low-molecular-weight non-amelogenin enamel polypeptides and their parent protein, which concentrate in the sheath space separating rod and inter-rod enamel (Uchida et al., 1995). Two porcine sheath proteins, with apparent molecular weights of 13 and 15 kDa, are characterized by protein sequencing. The primary structures of these polypeptides match a portion of the derived amino acid sequences of clones isolated from a porcine enamel organ epithelia-specific cDNA library. Sheath protein RNA messages differ by the inclusion or deletion of a 45-nucleotide segment and by the use of three alternative polyadenylation/cleavage sites. The secreted proteins are 395 and 380 residues in length, with molecular masses of 42,358 and 40,279 Daltons and calculated isoelectric points of 6.3 and 6.7, respectively. Polyclonal antibodies were raised against a synthetic peptide having the sheathlin-specific sequence EHETQQYEYSGGC. Immunohistochemistry with this antibody demonstrates that the protein encoded by the sheathlin cDNA is preferentially localized in the sheath space. We propose that the porcine sheath proteins and their proteolytic cleavage products be designated "sheathlin".

• Spicer AP, Duhig T, Chilton BS, Gendler SJ
• Analysis of mammalian MUC1 genes reveals potential functionally important domains.
• Mamm Genome. 1995; 6: 885-8

• Smith WC et al.
• Alligator rhodopsin: sequence and biochemical properties.
• Exp Eye Res. 1995; 61: 569-78
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• We sequenced selected peptides of alligator rhodopsin that accounted for about half of the total protein. These sequences were confirmed when the total primary structure of alligator rhodopsin was deduced from the cDNA sequence. Differences in the amino-terminal region, compared to that of bovine rhodopsin, account for failure of cross-reactivity of several anti-bovine rhodopsin monoclonal antibodies. Differences in the carboxyl-terminal region give rise to limited antibody cross-reactivity and may also account for a slightly reduced ability of alligator rhodopsin to be phosphorylated by bovine rhodopsin kinase. Alligator rhodopsin regenerates much faster than bovine rhodopsin. The pseudo-first-order rate constant for alligator rhodopsin regeneration is approximately 25 times that of bovine. Phylogenetic analysis of 17 rhodopsin sequences indicates that the alligator is more closely related to the chicken than to the other species examined.

• Amato G, Gatesy J
• PCR assays of variable nucleotide sites for identification of conservation units.
• EXS. 1994; 69: 215-26
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• A number of authors have recently suggested that the best approach for identifying units of conservation is to follow a systematics model of character analysis (Amato, 1991; Cracraft, 1991; Vogler and DeSalle, 1994). This approach necessitates the use of an operational, typological, evolutionary species concept. The use of the phylogenetic species concept has the utility and philosophical logic appropriate for this task. Additionally, there is a large body of literature that uses this framework, along with a parsimony based character analysis to identify patterns of phylogeny (Cracraft, 1983; Nelson and Platnick, 1981; Nixon and Wheeler, 1990). While we advocate this approach, we recognize that one of its limiting factors is sample size. We propose that by selective direct sequencing plus rapid sampling of variable target characters by polymerase chain reaction (PCR) assays of specific sites, sufficiently large numbers of individuals can be accurately, inexpensively, and quickly surveyed for diagnostic characters. This procedure is demonstrated by a survey of variable nucleotide sites in the Caiman crocodilus complex.

• Karlin S
• Statistical studies of biomolecular sequences: score-based methods.
• Philos Trans R Soc Lond B Biol Sci. 1994; 344: 391-402
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• The massive accumulation of DNA and protein sequence data poses challenges and opportunities in terms of interpretation and analysis. This presentation reviews the method of score-based sequence analysis with the objectives of discerning distinctive segments in single sequences and identifying significant common segments in sequence comparisons. A number of new results are described here for both the theory and its applications. These include distributional theory involving several high scoring segments in single sequences, distribution formulas for general scoring regimes in multiple sequence comparisons, bounds for periodic scoring assignments, sensitivity analysis of genome composition and refinements on predicting exons and genes in DNA sequences.

• Larhammar D, Milner RJ
• Phylogenetic relationship of birds with crocodiles and mammals, as deduced from protein sequences.
• Mol Biol Evol. 1989; 6: 693-6

• Gartside DF, Dessauer HC, Joanen T
• Genic homozygosity in an ancient reptile (Alligator mississippiensis).
• Biochem Genet. 1977; 15: 655-63
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• Proteins assayed electrophoretically showed variation at only three of 49 presumed genetic loci in alligators from southwestern Louisiana. Average heterozygosity per individual was 0.021+/-0.012; proportion of polymorphic loci was 0.06. Data on the history, structure, and ecology of this alligator population are consistent with natural selection as the primary factor accounting for this low genetic variability. However, neither a historic population bottleneck nor some genetic mechanism limiting variability can be dismissed as a possible factor.

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