Aspartic proteinase genes in the Brassicaceae Arabidopsis thaliana and Brassica napus

Active aspartic proteinase is isolated from Brassica napus seeds and the peptide sequence is used to generate primers for PCR. We present here cDNA and genomic clones for aspartic proteinases from the closely related Brassicaceae Arabidopsis thaliana and Brassica napus. The Arabidopsis cDNA represents a single gene, while Brassica has at least 4 genes. Like other plant aspartic proteases, the two Brassicaceae enzymes contain an extra protein domain of about 100 amino acids relative to the mammalian forms. The intron/exon arrangement in the Brassica genomic clone is significantly different from that in mammalian genes. As the proteinase is isolated from seeds, the same tissue where 2S albumins are processed, this implies expression of one of the aspartic proteinase genes there.     

Heterogeneity and Polymorphism of Functionally Specialized Blood Proteins in Migratory Fish: The Case Study of the North Caspian Population of the Russian Sturgeon during Sea and River Periods of Life. 1. Albumins

A comparative study of the levels of heterogeneity and polymorphism of albumins, the most important functionally specialized blood proteins, has been carried out. The albumin system of Russian sturgeon undergoes distinct changes while the fish change habitat during the spawning migration from sea to river. They are expressed as an increased level of heterogeneity, an increased content of serum albumins in fish during the river period of life as compared to the sea period, and an increased share of a slow component of albuminograms. These changes suggest a significant role of the blood albumin system in adaptation of the Caspian sturgeon migratory species to fresh water life conditions.     

Bisalbuminemia in an amphibian

Improved electrophoretic resolution revealed two albumin-like proteins in Taricha granulosa plasma (bisalbuminemia). The Taricha proteins were compared to mammalian, avian and reptilian serum albumins regarding molecular weight, amino acid composition, isoelectric character, solubility and the binding of hemin and dyes. The results indicate that although the two Taricha proteins have demonstrated hemoglobin-binding ability, they possess traits that characterize them to be true serum albumins.     

Age-related variability of serum protein antigens in sturgeons (Acipenseridae, Acipenseriformes)

We studied the variability of antigenic properties of proteins in two sturgeon species at different stages of postembryonic development. The deepest changes occurred in individual components of albumins and beta-globulins (transferrins) and were mostly related to an increased proportion of the protein accounting for these antigens. Transformation of the main component of albumins A1 into adult antigens was completed in 5-month old fry. The main component of beta-globulins A (component of transferrins) appeared in the blood flow much later than other proteins and could retain the fry features until the age of 3-4 years. Other antigens belonging to alpha1- and alpha2-globulins and the second component of transferrins were more stable and did not undergo substantial changes. The direction of ontogenetic variability of serum antigens in sturgeon fry did not depend on the habitat of adult fish in fresh or sea water.     

Cloning of fish enzymes and other fish protein genes

Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.     

Fluorescence-detected circular dichroism studies of serum albumins

Multidimensional fluorescence-detected circular dichroism (FDCD) is used to investigate the binding of bilirubin to four mammalian serum albumins. It is shown that the complexes formed with the albumins have distinctly different FDCD spectra. The effect of the pH-dependent transition from basic to acidic conditions on the complexes is examined. The binding of warfarin to albumin is also presented to demonstrate the general analytical utility of the multidimensional FDCD measurement for biochemical analysis.     

Suppressive Subtraction Libraries to Identify Interferon-Inducible Genes in Fish

Previous attempts to identify genes in fish that respond to virus infection or interferon induction have not been particularly productive. Since these genes are very important in developing strategies to control disease outbreaks in aquaculture, we began a study of interferon-inducible genes in fish using suppressive subtraction hybridization to construct cDNA libraries enriched for interferon-inducible genes. Subtraction hybridization libraries were constructed with cDNA obtained from the kidney, spleen, and liver of Chinook salmon (Oncorhynchus tshawytscha) and staghorn sculpin (Hemilepidotus spinosus) before and after injection with poly IC, a potent interferon inducer. The ``identified' genes in both cDNA libraries corresponded to previously identified genes of the fish complement system, the interferon-inducible proteins observed in mammalian cells, and the Vig-1 gene, identified in fish cells after infection with fish rhabdoviruses. Received June 19, 2001; accepted July 13, 2001     

Evolution of sarcomeric myosin heavy chain genes: evidence from fish

Myosin heavy chain (MYH) is a major structural protein, integral to the function of sarcomeric muscles. We investigated both exon-intron organization and amino acid sequence of sarcomeric MYH genes to infer their evolutionary history in vertebrates. Our results were consistent with the hypothesis that a multigene family encoded MYH proteins in the ancestral chordate, one gene ancestral to human MYH16 and its homologues and another ancestral to all other vertebrate sarcomeric MYH genes. We identified teleost homologues of mammalian skeletal and cardiac MYH genes, indicating that the ancestors of those genes were present before the divergence of actinopterygians and sarcopterygians. Indeed, the ancestral skeletal genes probably duplicated at least once before the divergence of teleosts and tetrapods. Fish homologues of mammalian skeletal MYH are expressed in skeletal tissue and homologues of mammalian cardiac genes are expressed in the heart but, unlike mammals, there is overlap between these expression domains. Our analyses inferred two other ancestral vertebrate MYH genes, giving rise to human MYH14 and MYH15 and their homologues. Relative to the skeletal and cardiac genes, MYH14 and MYH15 homologues are characterized by evolution of intron position, differences in evolutionary rate between the functionally differentiated head and rod of the myosin protein, and possible evolution of function among vertebrate classes. Tandem duplication and gene conversion appear to have played major roles in the evolution of at least cardiac and skeletal MYH genes in fish. One outcome of this high level of concerted evolution is that different fish taxa have different suites of MYH genes, i.e., true orthologs do not exist.     

Cloning of Fish Enzymes and Other Fish Protein Genes

ABSTRACT:?

Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.     

Terrestrial vertebrates have two keratin gene clusters; striking differences in teleost fish

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.     

鱼类干扰素反应及分子调控

干扰素反应在脊椎动物抵抗病毒感染过程中发挥重要作用。近年来,鱼类干扰素抗病毒免疫反应的研究取得了重要进展,不仅克隆鉴定了一系列鱼类干扰素系统基因,而且通过功能研究揭示了鱼类具有类似于高等哺乳类的干扰素反应。但是,鱼类干扰素反应及分子调控具有自身特点。以下综述了最近这方面的研究结果。     

Granule Associated Serine Proteases of Hematopoietic Cells – An Analysis of Their Appearance and Diversification during Vertebrate Evolution

Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.     

Serum albumins—Unusual allergens

Background

Albumins are multifunctional proteins present in the blood serum of animals. They can bind and transport a wide variety of ligands which they accommodate due to their conformational flexibility. Serum albumins are highly conserved both in amino acid sequence and three-dimensional structure. Several mammalian and avian serum albumins (SAs) are also allergens. Sensitization to one of the SAs coupled with the high degree of conservation between SAs may result in cross-reactive antibodies in allergic individuals. Sensitivity to SA generally begins with exposure to an aeroallergen, which can then lead to cross-sensitization to serum albumins present in food.

Scope of review

This review focuses on the allergenicity of SAs presented in a structural context.

Major conclusions

SA allergenicity is unusual taking into account the high sequence identity and similarity between SA from different species and human serum albumin. Cross-reactivity of human antibodies towards different SAs is one of the most important characteristics of these allergens.

General significance

Establishing a relationship between sequence and structure of different SAs and their interactions with antibodies is crucial for understanding the mechanisms of cross-sensitization of atopic individuals. Structural information can also lead to better design and production of recombinant SAs to replace natural proteins in allergy testing and desensitization. Therefore, structural analyses are important for diagnostic and treatment purposes. This article is part of a Special Issue entitled Serum Albumin.     

The interferon system of teleost fish

Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-lambda) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-alpha and IFN-beta, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-lambdas have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-gamma, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-alpha, -beta and -lambda groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-lambdas, but show most sequence similarity to IFN-alpha. Recently, IFN-gamma genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-gamma genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-alpha/beta-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK-STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated.     

Evolution of glucagon genes

Statistical analyses of DNA sequences of the preproglucagon genes from bovine, human, hamster, and anglerfish suggest that a gene duplication creating two anglerfish genes (AF I and II) occurred about 160 Myr ago, long after the separation of fish and mammals. The analyses further suggest that the internal duplication producing the glucagon and glucagon-like peptide II (GLP-II) regions occurred about 1.2 billion years ago, which would indicate that the GLP-II region was present in the ancestral anglerfish sequence but was silenced or deleted before the gene duplication separating AF I and II. The glucagon-like peptide I (GLP-I) was derived from a duplication of the ancestral glucagon region about 800 Myr ago. The rate of synonymous substitution in these genes is approximately 4.3 x 10(-9) substitutions per year per synonymous site. The rate of nonsynonymous substitution in the signal peptide region is about 1.1 x 10(-9) substitutions per year per nonsynonymous site, a high rate comparable to that in the C-peptide region of preproinsulin. The rate of nonsynonymous substitution in the glicentin-related pancreatic polypeptide (GRPP) region is 0.63 x 10(-9) for the comparisons between mammalian species and 1.8 x 10(-9) for the comparisons between fish and mammals; the moderate rate in mammals suggests a physiological role for GRPP. The glucagon region is extremely conservative; no nonsynonymous substitution is observed in the mammalian genes, and a nonsynonymous rate of 0.18 x 10(-9) was obtained from the comparisons between fish and mammals. In the GLP-I region, the rate of nonsynonymous substitution was estimated to be 0.08 x 10(-9) for the comparisons between mammalian species and 0.30 x 10(- 9) for the comparisons between fish and mammals. In the GLP-II region, the rate was estimated to be 0.25 x 10(-9) for the comparisons between mammalian species. Thus, GLP-I and II are also very conservative, which suggests an important physiological role for these peptides.      

Molecular Evolution of Vertebrate Goose-Type Lysozyme Genes

We have found that mammalian genomes contain two lysozyme g genes. To better understand the function of the lysozyme g genes we have examined the evolution of this small gene family. The lysozyme g gene structure has been largely conserved during vertebrate evolution, except at the 5' end of the gene, which varies in number of exons. The expression pattern of the lysozyme g gene varies between species. The fish lysozyme g sequences, unlike bird and mammalian lysozyme g sequences, do not predict a signal peptide, suggesting that the encoded proteins are not secreted. The fish sequences also do not conserve cysteine residues that generate disulfide bridges in the secreted bird enzymes, supporting the hypothesis that the fish enzymes have an intracellular function. The signal peptide found in bird and mammalian lysozyme g genes may have been acquired as an exon in the ancestor of birds and mammals, or, alternatively, an exon encoding the signal peptide has been lost in fish. Both explanations account for the change in gene structure between fish and tetrapods. The mammalian lysozyme g sequences were found to have evolved at an accelerated rate, and to have not perfectly conserved the known active site catalytic triad of the bird enzymes. This observation suggests that the mammalian enzymes may have altered their biological function, as well.     

Proteins of mammalian mitochondrial ribosomes.

The bovine mitochondrial system is being developed as a model system for studies on mammalian mitochondrial ribosomes. Information is emerging on the structural organization and RNA binding properties of proteins in these mitochondrial ribosomes. Unexpectedly, these ribosomes appear to interact directly with GTP, via a high affinity binding site on the small subunit. Despite major differences in their RNA content and physical properties, mammalian mitochondrial and cytoplasmic ribosomes contain about the same number of proteins. The proteins in each kind of ribosome have a similar size distribution, and both sets are entirely coded by nuclear genes, raising the possibility that these different ribosomes may contain the same set of proteins. Comparison of bovine mitochondrial and cytoplasmic r-proteins by co-electrophoresis in two-dimensional gels reveals that most of the cytoplasmic ribosomal proteins are more basic than the mitochondrial ribosomal proteins, and that none are co-migratory with mitochondrial ribosomal proteins, suggesting that the proteins in the two ribosomes are different. To exclude the possibility that the electrophoretic differences result only from post-translational modification of otherwise identical proteins, antibodies against several proteins from the large subunit of bovine mitochondrial ribosomes were tested against cytoplasmic ribosomes by solid phase radioimmunoassay and against cytoplasmic ribosomal proteins on Western blots. The lack of cross-reaction of these antibodies with cytoplasmic r-proteins suggests that mitochondrial ribosomal proteins have different primary structures and thus are most likely encoded by a separate set of nuclear genes.