RESUMEN
The cytoskeleton is crucial in determining cell architecture, division, motility, transport processes and in local control of signal transduction. Relatives of actin and tubulin are expressed in all phyla, underlining the fundamental importance of conserved cytoskeletal functions. Intermediate filament proteins have evolved in parallel with tissue diversity in the animal kingdom, likely from the demand to adapt one class of cytoskeletal proteins to cell type-restricted functions. Up to now, the evolutionary origin of cytoplasmic intermediate filament proteins remains unknown. Using a known gene encoding a cytoplasmic intermediate filament protein from the hemichordate Saccoglossus kowalevskii, we have identified the first corresponding gene in the sea anemone Nematostella, tentatively named cytovec. Our data reveal a relationship of cytovec with Hydra vulgaris nematocilins A and B that also lack a CAAX box. In light of additional recent findings, our data show that cytoplasmic intermediate filament genes are present in the common ancestor of Cnidaria and Bilateria.
Asunto(s)
Proteínas de Filamentos Intermediarios/genética , Anémonas de Mar/genética , Animales , Secuencia de Bases , Proteínas de Filamentos Intermediarios/química , Proteínas de Filamentos Intermediarios/aislamiento & purificación , Datos de Secuencia Molecular , Anémonas de Mar/química , Análisis de Secuencia de ADNRESUMEN
We collected as much information as possible on new lamin genes and their flanking genes. The number of lamin genes varies from 1 to 4 depending more or less on the phylogenetic position of the species. Strong genome drift is recognised by fewer and unusually placed introns and a change in flanking genes. This applies to the nematode Caenorhabditis elegans, the insect Drosophila melanogaster, the urochordate Ciona intestinalis, the annelid Capitella teleta and the planaria Schmidtea mediterranea. In contrast stable genomes show astonishing conservation of the flanking genes. These are identical in the sea anemone Nematostella vectensis and the cephalochordate Branchiostoma floridae lamin B1 gene. Even in the lamin B1 genes from Xenopus tropicalis and man one of the flanking genes is conserved. Finally our analysis forms the basis for a molecular analysis of metazoan phylogeny.
Asunto(s)
Evolución Molecular , Genes Esenciales/genética , Laminas/genética , Secuencia de Aminoácidos , Animales , Genoma , Intrones/genética , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
We identified the single gene for nuclear lamin in the genome draft of the sea anemone Nematostella vectensis, a member of the cnidaria, a very old metazoan phylum. The gene consists of 10 exons and 9 introns. Strikingly all 9 intron positions are conserved in the human lamin B genes, which have only 1 (lamin B1) or 2 (lamin B2) additional introns. Using the information on neighboring genes we propose that the human lamin B1 gene on chromosome 5 is the true homolog of the Nematostella lamin gene, while the lamin B2 gene on chromosome 19 arose during vertebrate evolution. In marked contrast to this conservation of gene structure are the results in the rapidly evolving genomes of Drosophila and Caenorhabditis elegans. Here the lamin genes have much fewer introns and these occur often at novel positions. In the single nematode lamin gene and the Drosophila lamin Dmo gene no intron position coincides with an intron in the sea anemone lamin gene.
Asunto(s)
Secuencia Conservada , Evolución Molecular , Intrones/genética , Laminas/genética , Anémonas de Mar/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Orden Génico , Humanos , Proteínas de Filamentos Intermediarios/genética , Lamina Tipo B/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de SecuenciaRESUMEN
We have recently demonstrated that the keratin K3 gene, which is active in the suprabasal human corneal epithelium, is missing in the genome of the mouse. We show that a normal K3 gene exists in a wide variety of mammals while in rodents the gene is converted to a pseudogene with a very strong sequence drift. The availability of K5-/- mice provides a unique opportunity to investigate type-specific keratin function during corneal differentiation in the absence of both K5 and K3. Here, we report that the deletion of K5, which in wild-type mice forms a cytoskeleton with K12, does neither cause keratin aggregation nor cytolysis in the cornea. This is due to the induction of K4 in corneal epithelial cells, normally restricted to corneal stem stem cells residing in the limbus. Using a combination of antibodies and RT-PCR, we identified additional keratins expressed in the mouse cornea including K23 which was previously thought to be specific for pancreatic carcinomas. This reflects an unexpected complexity of keratin expression in the cornea. Our data suggest that in the absence of mechanical stress, corneal differentiation does not depend on distinct keratin pairs, supporting a concept of functional redundancy, at least for certain keratins.
Asunto(s)
Epitelio Corneal/metabolismo , Expresión Génica/genética , Queratinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular/fisiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Epitelio Corneal/citología , Epitelio Corneal/ultraestructura , Humanos , Queratina-15 , Queratina-5 , Queratinas/genética , Queratinas/inmunología , Ratones , Ratones Endogámicos , Ratones Noqueados , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Datos de Secuencia Molecular , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
The genomic database for a marsupial, the opossum Monodelphis domestica, is highly advanced. This allowed a complete analysis of the keratin I and keratin II gene cluster with some 30 genes in each cluster as well as a comparison with the human keratin clusters. Human and marsupial keratin gene clusters have an astonishingly similar organization. As placental mammals and marsupials are sister groups a corresponding organization is also expected for the archetype mammal. Since hair is a mammalian acquisition the following features of the cluster refer to its origin. In both clusters hair keratin genes arose at an interior position. While we do not know from which epithelial keratin genes the first hair keratins type-I and -II genes evolved, subsequent gene duplications gave rise to a subdomain of the clusters with many neighboring hair keratin genes. A second subdomain accounts in both clusters for 4 neighboring genes encoding the keratins of the inner root sheath (irs) keratins. Finally the hair keratin gene subdomain in the type-I gene cluster is interrupted after the second gene by a region encoding numerous genes for the high/ultrahigh sulfur hair keratin-associated proteins (KAPs). We also propose a tentative synteny relation of opossum and human genes based on maximal sequence conservation of the encoded keratins. The keratin gene clusters of the opossum seem to lack pseudogenes and display a slightly increased number of genes. Opossum keratin genes are usually longer than their human counterparts and also show longer intergenic distances.
Asunto(s)
Mapeo Cromosómico , Genoma Humano , Queratinas/genética , Monodelphis/genética , Familia de Multigenes , Animales , Citoplasma/química , ADN/análisis , ADN/genética , Bases de Datos de Ácidos Nucleicos , Epitelio/química , Duplicación de Gen , Cabello/química , Humanos , Queratinas/análisisRESUMEN
Keratins I and II form the largest subgroups of mammalian intermediate filament (IF) proteins and account as obligatory heteropolymers for the keratin filaments of epithelia. All human type I genes except for the K18 gene are clustered on chromosome 17q21, while all type II genes form a cluster on chromosome 12q13, that ends with the type I gene K18. Highly related keratin gene clusters are found in rat and mouse. Since fish seem to lack a keratin II cluster we screened the recently established draft genomes of a bird (chicken) and an amphibian (Xenopus). The results show that keratin I and II gene clusters are a feature of all terrestrial vertebrates. Because hair with its multiple hair keratins and inner root sheath keratins is a mammalian acquisition, the keratin gene clusters of chicken and Xenopus tropicalis have only about half the number of genes found in mammals. Within the type I clusters all genes have the same orientation. In type II clusters there is a rare gene of opposite orientation. Finally we show that the genes for keratins 8 and 18, which are the first expression pair in embryology, are not only adjacent in mammals, but also in Xenopus and three different fish. Thus neighboring K8 and K18 genes seem a feature shared by all vertebrates. In contrast to the two well defined keratin gene clusters of terrestrial vertebrates, three teleost fish show an excess of type I over type II genes, the lack of a keratin type II gene cluster and a striking dispersal of type I genes, that are probably the result of the teleost-specific whole genome duplication followed by a massive gene loss. This raises the question whether keratin gene clusters extend beyond the ancestral bony vertebrate to cartilage fish and lamprey. We also analyzed the complement of non-keratin IF genes of the chicken. Surprisingly, an additional nuclear lamin gene, previously overlooked by cDNA cloning, is documented on chromosome 10. The two splice variants closely resemble the lamin LIII a + b of amphibia and fish. This lamin gene is lost on the mammalian lineage.
Asunto(s)
Anfibios/genética , Aves/genética , Peces/genética , Queratinas/genética , Familia de Multigenes , Animales , Pollos/genética , Mapeo Cromosómico , Humanos , Filamentos Intermedios/genética , Xenopus/genética , Pez Cebra/genéticaRESUMEN
Here, we present the comparative analysis of the two keratin (K) gene clusters in the genomes of man, mouse and rat. Overall, there is a remarkable but not perfect synteny among the clusters of the three mammalian species. The human type I keratin gene cluster consists of 27 genes and 4 pseudogenes, all in the same orientation. It is interrupted by a domain of multiple genes encoding keratin-associated proteins (KAPs). Cytokeratin, hair and inner root sheath keratin genes are grouped together in small subclusters, indicating that evolution occurred by duplication events. At the end of the rodent type I gene cluster, a novel gene related to K14 and K17 was identified, which is converted to a pseudogene in humans. The human type II cluster consists of 27 genes and 5 pseudogenes, most of which are arranged in the same orientation. Of the 26 type II murine keratin genes now known, the expression of two new genes was identified by RT-PCR. Kb20, the first gene in the cluster, was detected in lung tissue. Kb39, a new ortholog of K1, is expressed in certain stratified epithelia. It represents a candidate gene for those hyperkeratotic skin syndromes in which no K1 mutations were identified so far. Most remarkably, the human K3 gene which causes Meesmann's corneal dystrophy when mutated, lacks a counterpart in the mouse genome. While the human genome has 138 pseudogenes related to K8 and K18, the mouse and rat genomes contain only 4 and 6 such pseudogenes. Our results also provide the basis for a unified keratin nomenclature and for future functional studies.
Asunto(s)
Queratinas/genética , Familia de Multigenes , Animales , Secuencia Conservada , Bases de Datos como Asunto , Genoma , Genoma Humano , Humanos , Ratones , Modelos Genéticos , Mutación , Estructura Terciaria de Proteína , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la EspecieRESUMEN
We screened the recently established draft genome of the early chordate Ciona intestinalis for genes encoding cytoplasmic intermediate filament (IF) proteins. The draft of the tunicate/urochordate genome contains only the five genes (IF-A, IF-B, IF-C, IF-D and IF-F) previously established by cDNA cloning. Three of these IF proteins (IF-D, IF-C, IF-A) were shown to be orthologs of vertebrate IF subfamilies I to III while two proteins (IF-B, IF-F) seemed tunicate specific. This is now firmly established for protein IF-F since the genomic data show that it arises as a fusion protein with a C-terminal annexin domain, a feature not found before in the very large collection of metazoan IF proteins. The results also confirm the previous proposal that urochordates lack orthologs of vertebrate type IV IF proteins. We discuss the striking increase of IF complexity from 5 tunicate to 65 human genes during chordate evolution. Thus the tunicate has a single keratin pair, which is expressed in the epidermis, while the human genome has at least 25 genes each for keratins I and keratins II. Finally there are four normal Ciona annexin genes in addition to the gene encoding the IF-annexin fusion proteins (IF-F).
Asunto(s)
Anexinas/genética , Ciona intestinalis/genética , Genoma , Proteínas de Filamentos Intermediarios/genética , Filamentos Intermedios/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citoplasma/metabolismo , ADN Complementario/química , ADN Complementario/genética , Queratinas/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
We screened the genomic sequences of the teleost fish Fugu rubripes for genes that encode cytoplasmic intermediate filament (IF) proteins. Here, we compare the number of genes per subfamily (I to IV) as well as the gene mapping in the human and fish genomes. There are several unexpected differences. F. rubripes has a sizeable excess of keratin type I genes over keratin type II genes. Four of the six keratin type II genes map close to four keratin type I genes. Thus, a single keratin II gene cluster (as in mammals) seems excluded. Although a continuous genome sequence is not yet available for F. rubripes, it is difficult to see how all 19 keratin type I genes can be collected as in the human genome into a single cluster without the presence of type II genes and various unrelated genes. F. rubripes has more type III and type IV genes than humans. Some of the type IV genes acquired additional novel intron positions. One gene even harbors (in addition to the two type IV introns) three novel introns and three introns usually present only in mammalian and F. rubripes type I-III genes. This mixture of type IV and type I-III intron positions poses a problem for the traditional view that the first type IV gene arose in evolution by a mRNA-mediated translocation event. In the 42 F. rubripes genes analysed here, there are several differences in intron patterns compared with mammalian genes. Most correspond to additional introns in the fish genes. A search for genes encoding nuclear lamins reveals the four established fish lamins (A, B1, B2 and LIII) as well as an unexpected second lamin A.
Asunto(s)
Genoma Humano , Proteínas de Filamentos Intermediarios/genética , Takifugu/genética , Secuencia de Aminoácidos , Animales , Humanos , Intrones/genética , Queratinas/genética , Lamina Tipo A/genética , Datos de Secuencia Molecular , Proteínas de Neurofilamentos/genética , Especificidad de la EspecieRESUMEN
The urochordate Ciona intestinalis is a well established system for embryological studies, and large scale EST sequences begin to emerge. We cloned five cytoplasmic intennediate filament (IF) cDNAs and made specific antibodies to the recombinant proteins. Self-assembly studies and immunofluorescence microscopy were used to study these proteins and their distribution. Confirming and extending previous studies in Styela, we found that Ciona protein IF-A is expressed in muscle and forms homopolymeric filaments while proteins IF-C and IF-D, which form only obligatory heteropolymeric filaments, resemble a keratin pair exclusively found in the entire epidermis. Protein IF-B and the new protein IF-F potentially reflect tunicate-specific IF proteins. They are found in the entire internal epithelia including the neural gland. We also extended the analysis to earlier developmental stages of Ciona. Protein IF-A is expressed in muscle from larval stages, whereas proteins IF-C and IF-D are found only in the tail epidermis. Protein IF-F is detected abundantly in the test cells of eggs, embryos and premetamorphic larvae. Our studies show that IF proteins could prove very useful markers in the study of cell fate determination in Ciona. They also support previous findings on the evolutionary relationships of different IF proteins. Non-vertebrate chordates have IF proteins which represent orthologs of vertebrate type I to III proteins, but also IF proteins that do not seem to fit into these classes. However, the intron positions of all tunicate IF genes are conserved with vertebrate type I to III genes, pointing to a common evolutionary origin.
Asunto(s)
Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Urocordados/embriología , Urocordados/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Biomarcadores , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/química , Proteínas de Filamentos Intermediarios/clasificación , Filamentos Intermedios/química , Filamentos Intermedios/metabolismo , Filamentos Intermedios/ultraestructura , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Urocordados/citología , Urocordados/genéticaRESUMEN
We have isolated full length cDNAs encoding a cytoplasmic intermediate filament (IF) protein of a priapulid (Priapulus caudatus) and of a hemichordate (Saccoglossus kowalevskii) and determined the organisation of the hemichordate gene. As expected, the priapulid protein shows the long coil 1b subdomain and the lamin tail homology segment already known for cytoplasmic IF proteins from 11 other protostomic phyla. Surprisingly, the hemichordate protein follows in domain organisation much more closely the protostomic IF proteins than the chordate IF proteins. Thus, the lack of a lamin tail homology segment together with a deletion of 42 residues in the coil 1b domain is a molecular feature restricted to the chordates. We propose that the known IF subfamilies I to IV developed by gene duplications and sequence drift after the deletion in coil 1b arose at the base of the chordate branch.