Identification and Comparative Analysis of the Protocadherin Cluster in a Reptile,the Green Anole Lizard

Background

The vertebrate protocadherins are a subfamily of cell adhesion molecules that are predominantly expressed in the nervous system and are believed to play an important role in establishing the complex neural network during animal development. Genes encoding these molecules are organized into a cluster in the genome. Comparative analysis of the protocadherin subcluster organization and gene arrangements in different vertebrates has provided interesting insights into the history of vertebrate genome evolution. Among tetrapods, protocadherin clusters have been fully characterized only in mammals. In this study, we report the identification and comparative analysis of the protocadherin cluster in a reptile, the green anole lizard (Anolis carolinensis).

Methodology/Principal Findings

We show that the anole protocadherin cluster spans over a megabase and encodes a total of 71 genes. The number of genes in the anole protocadherin cluster is significantly higher than that in the coelacanth (49 genes) and mammalian (54–59 genes) clusters. The anole protocadherin genes are organized into four subclusters: the δ, α, β and γ. This subcluster organization is identical to that of the coelacanth protocadherin cluster, but differs from the mammalian clusters which lack the δ subcluster. The gene number expansion in the anole protocadherin cluster is largely due to the extensive gene duplication in the γb subgroup. Similar to coelacanth and elephant shark protocadherin genes, the anole protocadherin genes have experienced a low frequency of gene conversion.

Conclusions/Significance

Our results suggest that similar to the protocadherin clusters in other vertebrates, the evolution of anole protocadherin cluster is driven mainly by lineage-specific gene duplications and degeneration. Our analysis also shows that loss of the protocadherin δ subcluster in the mammalian lineage occurred after the divergence of mammals and reptiles. We present a model for the evolutionary history of the protocadherin cluster in tetrapods.     

Regulation of protocadherin gene expression by multiple neuron-restrictive silencer elements scattered in the gene cluster

The clustered protocadherins are a subfamily of neuronal cell adhesion molecules that play an important role in development of the nervous systems in vertebrates. The clustered protocadherin genes exhibit complex expression patterns in the central nervous system. In this study, we have investigated the molecular mechanism underlying neuronal expression of protocadherin genes using the protocadherin gene cluster in fugu as a model. By in silico prediction, we identified multiple neuron-restrictive silencer elements (NRSEs) scattered in the fugu protocadherin cluster and demonstrated that these elements bind specifically to NRSF/REST in vitro and in vivo. By using a transgenic Xenopus approach, we show that these NRSEs regulate neuronal specificity of protocadherin promoters by suppressing their activity in non-neuronal tissues. We provide evidence that protocadherin genes that do not contain an NRSE in their 5′ intergenic region are regulated by NRSEs in the regulatory region of their neighboring genes. We also show that protocadherin clusters in other vertebrates such as elephant shark, zebrafish, coelacanth, lizard, mouse and human, contain different sets of multiple NRSEs. Taken together, our data suggest that the neuronal specificity of protocadherin cluster genes in vertebrates is regulated by the NRSE-NRSF/REST system.     

Clustered protocadherin family

The brain is a complex system composed of enormous numbers of differentiated neurons, and brain structure and function differs among vertebrates. To examine the molecular mechanisms underlying brain structure and function, it is important to identify the molecules involved in generating neural diversity and organization. The clustered protocadherin (Pcdh) family is the largest subgroup of the diverse cadherin superfamily. The clustered Pcdh proteins are predominantly expressed in the brain and their gene structures in vertebrates are diversified. In mammals, the clustered Pcdh family consists of three gene clusters: Pcdh -α, Pcdh -β, and Pcdh -γ. During brain development, this family is upregulated by neuronal differentiation, and Pcdh-α is then dramatically downregulated by myelination. Clustered Pcdh expression continues in the olfactory bulb, hippocampus, and cerebellum until adulthood. Structural analysis of the first cadherin domain of the Pcdh-α protein revealed it lacks the features that classical cadherins require for homophilic adhesiveness, but it contains Pcdh-specific loop structures. In Pcdh-α, an RGD motif on a specific loop structure binds β1-integrin. For gene expression, the gene clusters are regulated by multiple promoters and alternative cis splicing. At the single-cell level, several dozen Pcdh -α and -γ mRNA are regulated monoallelically, resulting in the combinatorial expression of distinct variable exons. The Pcdh-α and Pcdh-γ proteins also form oligomers, further increasing the molecular diversity at the cell surface. Thus, the unique features of the clustered Pcdh family may provide the molecular basis for generating individual cellular diversity and the complex neural circuitry of the brain.     

Protocadherins

Protocadherins constitute the largest subgroup within the cadherin family of calcium-dependent cell-cell adhesion molecules. Recent progress in genome sequencing has enabled a refined phylogenetic analysis of protocadherins and led to the discovery of three large protocadherin clusters on human chromosome 5/mouse chromosome 18. Interestingly, many of the circa 70 protocadherins in mammals are highly expressed in the central nervous system. Roles in tissue morphogenesis and formation of neuronal circuits during early vertebrate development have been inferred. In the postnatal brain, protocadherins are possibly involved in the modulation of synaptic transmission and the generation of specific synaptic connections.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits fromSpinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Protocadherin 2C: a new member of the protocadherin 2 subfamily expressed in a redundant manner with OL-protocadherin in the developing brain.

Using cDNA of human protocadherin 2A (pc2A; originally known as protocadherin 2) as a probe, we cloned a new member of the protocadherin 2 subfamily from mouse brain cDNA libraries and named it protocadherin 2C (pc2C). It was similar to pc2A throughout its entire coding region, and its C-terminal region was highly conserved. The locus of the pc2C gene was on the mouse chromosome 18C where the pc2A gene is located, suggesting that genes of the pc2 subfamily form a gene cluster. The expression of pc2C was restricted to the nervous system, and the expression started in the embryonic stage and increased up to the adult stage. The expression pattern was quite similar to that of OL-protocadherin, a distinct class of protocadherin, although the timing and relative strength of expression were different. These results suggest that pc2C may be involved in neural development along with other classes of protocadherins.     

Molecular evolution of hemoglobins of antarctic fishes (Notothenioidei)

Amino acid sequences of α- and β-chains of human hemoglobin and of hemoglobins of coelacanth and 24 teleost fish species, including 11 antarctic and two temperate Notothenioidei, were analyzed using maximum parsimony. Trees were derived for the α- and β-chains separately and for tandemly arranged sequences, using the human and coelacanth sequences as outgroups in all analyses. The topologies of the trees of the α-and β-chains are highly congruent and indicate a specific pattern of gene duplications and gene expression of teleost hemoglobins which has not yet been investigated into more detail. The Notothenioid fish generally contain a single major hemoglobin and often a second minor component. The α- and β-chains of the major components form a monophyletic group in all investigated trees, with the nonantarctic Pseudaphritis as their sister taxon. The minor chains also are a monophyletic group and form an unresolved cluster with the major chains and the hemoglobins of tuna and red gurnard. The Notothenioid families Nototheniidae and Bathydraconidae appear to be paraphyletic. Received: 26 March 1997 / Accepted: 7 May 1997     

Recent progress in protocadherin research

Protocadherins constitute a large family belonging to the cadherin superfamily and function in different tissues of a wide variety of multicellular organisms. Protocadherins have unique features that are not found in classic cadherins. Expression of protocadherins is spatiotemporally regulated and they are localized at synapses in the CNS. Although protocadherins have Ca(2+)-dependent homophilic interaction activity, the activities are relatively weak. Some protocadherins have heterophilic interaction activity and the cytoplasmic domains associate with the unique cytoplasmic proteins, which are essential for their biological functions. Given the characteristic properties, the large size, and the diversity of members of the protocadherin family, protocadherins may participate in various biological processes. In particular, protocadherins seem to play a central role(s) in the CNS as related to synaptic function.     

A systematic study of fundamentals in α-helical coiled coil mimicry by alternating sequences of β- and γ-amino acids

Aimed at understanding the crucially important structural features for the integrity of α-helical mimicry by βγ-sequences, an α-amino acid sequence in a native peptide was substituted by differently arranged βγ-sequences. The self- and hetero-assembly of a series of αβγ-chimeric sequences based on a 33-residue GCN4-derived peptide was investigated by means of molecular dynamics, circular dichroism, and a disulfide exchange assay. Despite the native-like behavior of βγ alternating sequences such as retention of α-helix dipole and the formation of 13-membered α-helix turns, the αβγ-chimeras with different βγ substitution patterns do not equally mimic the structural behavior of the native parent peptide in solution. The preservation of the key residue contacts such as van der Waals interactions and intrahelical H-bonding, which can be met only by particular substitution patterns, thermodynamically favor the adoption of coiled coil folding motif. In this study, we show how successfully the destabilizing structural consequences of α → βγ modification can be harnessed by reducing the solvent-exposed hydrophobic surface area and placing of suitably long and bulky helix-forming side chains at the hydrophobic core. The pairing of αβγ-chimeric sequences with the native wild-type are thermodynamically allowed in the case of ideal arrangement of β- and γ-residues. This indicates a similarity in local side chain packing of β- and γ-amino acids at the helical interface of αβγ-chimeras and the native α-peptide. Consequently, the backbone extended residues are able to participate in classical “knob-into-hole” packing with native α-peptide.     

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

Background  

Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation.     

The mouse adducin gene family: alternative splicing and chromosomal localization

Mouse cDNA sequences encoding α, β, and γ adducins were cloned from a mouse reticulocyte cDNA library. The purified clones contain alternatively spliced exons from all three adducin genes. In the case of α and β, the inclusion of the alternatively spliced exons results in truncated polypeptide isoforms (called α-2 and β-2). The mouse predicted amino acid sequences are compared with published rat and human sequences. For completion of this comparison, cDNA encoding the rat β-1 carboxy terminus was cloned by PCR. The carboxy terminal region containing MARCKS homology, calmodulin-binding region-2, and spectrin-actin-binding site, is conserved among α-1, β-1, and γ-1 isoforms in mouse, rat, and humans. We also report here the localization of the gene encoding γ adducin (Add3) to murine Chr 19, in a region that shows conserved synteny with human Chr 10. Received: 1 June 1999 / Accepted: 25 August 1999     

Identification of FeS clusters in the glycyl-radical enzyme benzylsuccinate synthase via EPR and M?ssbauer spectroscopy

The anaerobic degradation pathway of toluene is initiated by the addition of the methyl group of toluene to the double bond of fumarate. This reaction is catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase (BSS). The enzyme consists of three subunits, α, β, and γ, and differs from most other glycyl-radical enzymes in having additional cofactors. We have purified a Strep-tagged nonactivated BSS from recombinant Escherichia coli and identified the additional cofactors as FeS clusters by UV/vis, EPR, and M?ssbauer spectroscopy. Analysis of the metal content as well as the EPR and M?ssbauer spectra indicated that BSS contains magnetically coupled low-potential [4Fe–4S] clusters. Several enzyme preparations showed differing amounts of [3Fe–4S] clusters that could be reconstituted to [4Fe–4S] clusters, indicating that they arise from partial decay of the initial [4Fe–4S] clusters. The most likely location of these FeS clusters in the enzyme are the small β and γ subunits, which are unique for the BSS subfamily of glycyl-radical enzymes and contain conserved cysteines as potential ligands.     

Karyopherin α7 (KPNA7), a divergent member of the importin α family of nuclear import receptors

Background  

Classical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin α and importin β. NLS cargo is recognized by importin α, which is bound by importin β. Importin β mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin β triggers disassembly of the complex. To date, six importin α family members, encoded by separate genes, have been described in humans.     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes

 The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-γ (IFN-γ)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 α- and β-subunits. The deduced amino acid sequences of the α- and β-subunits were 49% identical. We also determined the primary structure of the mouse PA28 γ-subunit (Ki antigen), a protein of unknown function structurally related to the α- and β-subunits. The amino acid sequence identity of the γ-subunit to the α- and β-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the α- and β-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-γ-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the γ-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a γ-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-γ-inducible α- and β-subunits emerged by gene duplication from a γ-subunit-like precursor. Received: 11 March 1997     

Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes

In human intestinal epithelial crypt (HIEC) cells, the PI3-K/Akt-1 pathway is crucial for the promotion of cell survival and suppression of anoikis. Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3-K isoforms in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src-mediated suppression of anoikis. We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent). Hence, HIEC cells selectively express PI3-K isoform complexes, translating into distinct roles in Akt-1 activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src-mediated suppression of anoikis.