Expression of cloned genes in pro- and eukaryotic cells

Expression of cloned genes in new environment is reviewed. Gene expression is possible under control of their own regulatory elements in the cells of related organisms. Genes may also function in cells of taxonomically remote organisms; for example, the genes of lower eukaryotes are active in bacterial cells, the Drosophila gene -- in yeast cells. The main principles of construction of pro- and eukaryotic vectore capable to provide the expression of DNA sequences in corresponding recipient cells are discussed.     

Expression of eukaryotic genes in Escherichia coli cells]

The paper presents a survey of literature concerned with the possibility of expression of plasmid-clones genes from eukaryotic organisms in bacteria cells. Studies on bacterial synthesis of somatostatin, human insulin, hormone of rat growth and proteins: chicken ovalbumin and mouse dihydrofolate reductase are discussed.     

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG1 or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.     

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5′-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.     

Long-branch attraction and the rDNA model of early eukaryotic evolution

Phylogenetic analyses of ribosomal RNA genes have become widely accepted as a framework for understanding broad-scale eukaryotic evolution. Nevertheless, conflicts exist between the phylogenetic placement of certain taxa in rDNA trees and their expected position based on fossils, cytology, or protein-encoding gene sequences. For example, pelobiont amoebae appear to be an ancient group based on cytologic features, but they are not among the early eukaryotic brances in rDNA analyses. In this report, the derived position of pelobionts in rDNA trees is shown to be unreliable and likely due to long-branch attraction among more deeply branching sequences. All sequences that branch near the base of the tree suffer from relatively high apparent substitution rates and exhibit greater variation in ssu rDNA sequence length. Moreover, the order of the branches leading from the root of the eukaryotic tree to the base of the so-called "crown taxa" is consistent with a sequential attachment, due to "long-branch" effects, of sequences with increasing rates of evolution. These results suggest that the basal eurkaryotic topology drawn from rDNA analyses may be, in reality, an artifact of variation in the rate of molecular evolution among eukaryotic taxa.     

Computational prediction of eukaryotic protein-coding genes

The human genome sequence is the book of our life. Buried in this large volume are our genes, which are scattered as small DNA fragments throughout the genome and comprise a small percentage of the total text. Finding these indistinct 'needles' in a vast genomic 'haystack' can be extremely challenging. In response to this challenge, computational prediction approaches have proliferated in recent years that predict the location and structure of genes. Here, I discuss these approaches and explain why they have become essential for the analyses of newly sequenced genomes.     

Telomeric location of Giardia rDNA genes.

Giardia lamblia telomeres have been isolated from a library enriched for repaired chromosome ends by (i) screening with a Plasmodium falciparum telomere and (ii) differential hybridization with Bal 31-digested and total G. lamblia DNA. Analysis of three clones isolated by this strategy has identified multiple tandem repeats of the 5-mer TAGGG. An oligonucleotide containing these repeats recognizes Bal 31-sensitive bands in Southern hybridizations and detects all G. lamblia chromosomes in pulsed-field gel electrophoresis separations. An abrupt transition from the G. lamblia rDNA sequence to telomeric repeats has been found in all three clones. In two of the clones the transition occurs at the same site, near the beginning of the large subunit rDNA sequence. In the third clone the transition occurs at a site in the intergenic spacer sequence between the rDNA genes. Hybridization of an rDNA probe to a pulsed-field separation of G. lamblia chromosomes indicates that rDNA genes are present on several chromosomes but vary in location from isolate to isolate. These results suggest that rRNA genes are clustered at telomeric locations in G. lamblia and that these clusters are mobile.     

Evolution of the intron-exon structure of eukaryotic genes

The origin and evolution of intron-exon structures continue to be controversial topics. Two alternative theories, the ‘exon theory of genes’ and the ‘insertional theory of introns’, debate the presence or absence of introns in primordial genes. Both sides of the argument have focused on the positions of introns with respect to protein and gene structures. A new approach has emerged in the study of the evolution of intron-exon structures: a population analysis of genes. One example is the statistical analysis of intron phases — the position of introns within or between codons. This analysis detected a significant signal of exon shuffling in the DNA sequence database containing both ancient and modern exon sequences: intron phase correlations, that is, the association together within genes of introns of the same phase. The results of this analysis suggest that exon shuffling played an important role in the origin of both ancient and modern genes.     

The spread of LAGLIDADG homing endonuclease genes in rDNA

Group I introns that encode homing endonuclease genes (HEGs) are highly invasive genetic elements. Their movement into a homologous position in an intron-less allele is termed homing. Although the mechanism of homing is well understood, the evolutionary relationship between HEGs and their intron partners remains unclear. Here we have focused on the largest family of HEGs (encoding the protein motif, LAGLIDADG) to understand how HEGs and introns move in rDNA. Our analysis shows the phylogenetic clustering of HEGs that encode a single copy of the LAGLIDADG motif in neighboring, but often evolutionarily distantly related, group I introns. These endonucleases appear to have inserted into existing introns independent of ribozymes. In contrast, our data support a common evolutionary history for a large family of heterologous introns that encode HEGs with a duplicated LAGLIDADG motif. This finding suggests that intron/double-motif HEG elements can move into heterologous sites as a unit. Our data also suggest that a subset of the double-motif HEGs in rDNA originated from the duplication and fusion of a single-motif HEG encoded by present-day ribozymes in LSU rDNA.     

Regularities of context-dependent codon bias in eukaryotic genes

Nucleotides surrounding a codon influence the choice of this particular codon from among the group of possible synonymous codons. The strongest influence on codon usage arises from the nucleotide immediately following the codon and is known as the N₁ context. We studied the relative abundance of codons with N₁ contexts in genes from four eukaryotes for which the entire genomes have been sequenced: Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. For all the studied organisms it was found that 90% of the codons have a statistically significant N₁ context-dependent codon bias. The relative abundance of each codon with an N₁ context was compared with the relative abundance of the same 4mer oligonucleotide in the whole genome. This comparison showed that in about half of all cases the context-dependent codon bias could not be explained by the sequence composition of the genome. Ranking statistics were applied to compare context-dependent codon biases for codons from different synonymous groups. We found regularities in N₁ context-dependent codon bias with respect to the codon nucleotide composition. Codons with the same nucleotides in the second and third positions and the same N₁ context have a statistically significant correlation of their relative abundances.     

Expression of catalytic antibodies in eukaryotic systems

Expression of recombinant antibodies in mammalian cells is one of the key problems in immuno-biotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively performed in yeast cells. We obtained expression strains of the methylotrophic yeast Pichia pastoris producing single-chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab), and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant K _d and the first order constant (k ₂) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and to the single-chain antibody A.17 expressed in CHO and E. coli cells, respectively.     

Y chromosomal fertility genes of Drosophila: a new type of eukaryotic genes

The Y chromosomal fertility genes of Drosophila are required for sperm differentiation. They are active only in primary spermatocytes where they form giant lampbrush loops. The molecular structure of these genes was investigated and revealed an unusual composition of DNA. Short, tandemly repeated sequence clusters are interrupted by longer and more heterogeneous sequences, which probably all represent transposable elements. No indication of the presence of protein-coding regions has been found within the fertility genes. However, the lampbrush loops bind site-specific proteins recognized by immunofluorescence techniques. This, together with other experimental data, led to the hypothesis that the Y chromosomal genes have a function in binding chromosomal proteins. The data and arguments in support of this gene model are summarized in this paper.     

Analysis of evolution of exon-intron structure of eukaryotic genes

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.     

Cloning of eukaryotic genes in single-strand phage vectors: the human interferon genes

Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-α) and fibroblast (HuIFN-β) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-α clone, 201, has a nucleotide sequence different from published HuIFN-α sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-α in Escherichia coli.