Inverse Complement Research Articles

Modular Polymerase Synthesis and Internal Protein Domain Swapping via Dual Opposed Frameshifts in the Ebola Virus L Gene.

Sequence analysis of the Zaire ebolavirus (EBOV) polymerase (L gene) mRNA, using online tools, identified a highly ranked -1 programmed ribosomal frameshift (FS) signal including an ideal slippery sequence heptamer (UUUAAAA), with an overlapping coding region featuring two tandem UGA codons, immediately followed by an RNA region that is the inverse complement (antisense) to a region of the mRNA of the selenoprotein iodothyronine deiodinase II (DIO2). This antisense interaction was confirmed in vitro via electrophoretic gel shift assay, using cDNAs at the EBOV and DIO2 segments. The formation of a duplex between the two mRNAs could trigger the ribosomal frameshift, by mimicking the enhancing role of a pseudoknot structure, while providing access to the selenocysteine insertion sequence (SECIS) element contained in the DIO2 mRNA. This process would allow the -1 frame UGA codons to be recoded as selenocysteine, forming part of a C-terminal module in a low abundance truncated isoform of the viral polymerase, potentially functioning in a redox role. Remarkably, 90 bases downstream of the -1 FS site, an active +1 FS site can be demonstrated, which, via a return to the zero frame, would enable the attachment of the entire C-terminal of the polymerase protein. Using a construct with upstream and downstream reporter genes, spanning a wildtype or mutated viral insert, we show significant +1 ribosomal frameshifting at this site. Acting singly or together, frameshifting at these sites (both of which are highly conserved in EBOV strains) could enable the expression of several modified isoforms of the polymerase. The 3D modeling of the predicted EBOV polymerase FS variants using the AI tool, AlphaFold, reveals a peroxiredoxin-like active site with arginine and threonine residues adjacent to a putative UGA-encoded selenocysteine, located on the back of the polymerase "hand". This module could serve to protect the viral RNA from peroxidative damage.

Unit-regularity of elements in rings

An element [Formula: see text] in a unital ring [Formula: see text] is said to have an inverse complement [Formula: see text] if [Formula: see text] is a unit of [Formula: see text] and [Formula: see text]. Unit-regular elements are studied from the viewpoint of the existence of inverse complements. As a source of unit-regular elements, we prove that if [Formula: see text] is a completely reducible submodule of [Formula: see text], then every element of [Formula: see text] is unit-regular if and only if any nonzero submodule of [Formula: see text] is not square zero. This generalizes some results due to Stopar in 2020. Finally, extending the case of real or complex matrices to the context of rings, we characterize the outer and reflexive inverses of a given unit-regular element depending only on its inverse complement.

Inverse complements and strongly unit regular elements

In this paper, the notion of “strongly unit regular element”, for which every reflexive generalized inverse is associated with an inverse complement, is introduced. Noting that every strongly unit regular element is unit regular, some characterizations of unit regular elements are obtained in terms of inverse complements and with the help of minus partial order. Unit generalized inverses of given unit regular element are characterized as sum of reflexive generalized inverses and the generators of its annihilators. Surprisingly, it has been observed that the class of strongly regular elements and unit regular elements are the same. Also, several classes of generalized inverses are characterized in terms of inverse complements.

Inverse complements of a matrix and applications

In this paper, the concept of “Inverse Complemented Matrix Method”, introduced by Eagambaram (2018), has been reestablished with the help of minus partial order and several new properties of complementary matrices and the inverse of complemented matrix are discovered. Class of generalized inverses and outer inverses of given matrix are characterized by identifying appropriate inverse complement. Further, in continuation, we provide a condition equivalent to the regularity condition for a matrix to have unique shorted matrix in terms of inverse complemented matrix. Also, an expression for shorted matrix in terms of inverse complemented matrix is given.

Modular knowledge systems accelerate human migration in asymmetric random environments.

Migration is a key mechanism for expansion of communities. In spatially heterogeneous environments, rapidly gaining knowledge about the local environment is key to the evolutionary success of a migrating population. For historical human migration, environmental heterogeneity was naturally asymmetric in the north-south (NS) and east-west (EW) directions. We here consider the human migration process in the Americas, modelled as random, asymmetric, modularly correlated environments. Knowledge about the environments determines the fitness of each individual. We present a phase diagram for asymmetry of migration as a function of carrying capacity and fitness threshold. We find that the speed of migration is proportional to the inverse complement of the spatial environmental gradient, and in particular, we find that NS migration rates are lower than EW migration rates when the environmental gradient is higher in the NS direction. Communication of knowledge between individuals can help to spread beneficial knowledge within the population. The speed of migration increases when communication transmits pieces of knowledge that contribute in a modular way to the fitness of individuals. The results for the dependence of migration rate on asymmetry and modularity are consistent with existing archaeological observations. The results for asymmetry of genetic divergence are consistent with patterns of human gene flow.

Analysis of the translational initiation region on the Euglena gracilis chloroplast ribulose-bisphosphate carboxylase/oxygenase (rbcL) messenger RNA.

The chloroplast mRNAs from Euglena gracilis fall into two classes. One group of mRNs from this organelle contains a Shine-Dalgarno sequence 5' to the start codon, while the other group of mRNAs does not have a conserved sequence signal in the 5'-untranslated region. To investigate the start signals for E. gracilis chloroplast mRNAs that do not carry a Shine-Dalgarno sequence, 30 S initiation complex formation has been studied using a series of transcripts carrying the wild-type translational start site of ribulose-bisphosphate carboxylase/oxygenase (rbcL) or mutated derivatives of this site. Mutation of the start codon of the rbcL gene indicates that the chloroplast 30 S subunit is recognizing only the correct AUG codon. The analysis of the messages from a series of deletion mutants shows that a minimum of delta 20 residues 5' to the AUG codon is required for activity. Maximal activity requires the full 55-base leader sequence. Surprisingly, a transcript carrying the inverse complement of 48 bases in the leader is delta 60% as active as the wild-type message in promoting initiation complex formation. Introduction of a Shine-Dalgarno sequence in the 5'-leader increases the activity of the mRNA only delta 1.4-2-fold. The presence of an oligodeoxynucleotide containing a strong Shine-Dalgarno sequence does not significantly inhibit the formation of initiation complexes at the rbcL start site. Similar results are obtained when initiation complexes are formed with initiation factors from either E. gracilis chloroplasts or Escherichia coli.

Derivation of the complete msp4 gene sequence of Anaplasma marginale without cloning

The gene encoding MSP4, a 31-kDa surface protein of the rickettsia Anaplasma marginale, was completely sequenced without prior cloning of the gene. Degenerate oligodeoxyribonucleotides (oligos) corresponding to the N-terminal amino-acid sequence of MSP4 were used as primers in the polymerase chain reaction (PCR) to amplify a 65-bp fragment of which the central 32 bp was nondegenerate. The 32-bp probe was hybridized to restriction enzyme-digested, genomic A. marginale DNA in Southern blots, and the hybridizing fragment was size selected from agarose gels and ligated into the Bluescript II SK (+/−) plasmid. The ligation reaction mixture was used as a template for PCR, amplified from the 32-bp oligo or its inverse complement to plasmid sequences. The PCR products were sequenced to obtain the entire msp4 gene and surrounding regions. This method of sequencing a gene without previously obtaining a clone for that gene could be beneficial in situations where conventional cloning and screening strategies are inefficient or ineffective.

Insertion of a 5' truncated L1 element into the 3' end of exon 44 of the dystrophin gene resulted in skipping of the exon during splicing in a case of Duchenne muscular dystrophy.

We report here the second evidence of retrotransposition of L1, which was found inserted into the dystrophin gene of a patient, causing Duchenne muscular dystrophy (DMD). When the PCR was used to amplify a region of the dystrophin gene encompassing exon 44 from genomic DNA of two Japanese brothers with DMD, it was found to be approximately 600 bp larger than expected. Both the normal and the abnormally large products were amplified from the DNA of their mother. However, the maternal grandparents did not have the abnormal allele, and the mutation must therefore have occurred in the mother. Analysis of nucleotide sequence of the amplified product from a patient disclosed that the insertion was present zero to two bases upstream from the 3' end of exon 44 and that two to four bases of the exon sequence were deleted from the insertion site. The insertion sequence was found to be composed of 606-608 bp and to be almost identical to the inverse complement of 3' portion of the L1 retrotransposon consensus sequence. The dystrophin gene transcript from peripheral lymphocytes of one of the patients was analyzed by using reverse transcription/semi-nested PCR. The size of the amplified product encompassing exon 42 to 46 was smaller than expected. Sequencing of the amplified product disclosed that the sequence of exon 43 was directly joined to that of exon 45. Exon 44 of the transcript was thus shown to be skipped during splicing. This novel mutation of the dystrophin gene has important implications regarding retrotransposition of an active L1 element and provides a new insight into the origins of mutations in the dystrophin gene.

A general method of polymerase-chain-reaction-enabled protein domain mutagenesis: Construction of a human protein S-osteonectin gene

Polymerase chain reaction (PCR) amplification was employed to construct a mosaic gene consisting of the propeptide region of protein S and the glutamic acid-rich domain of osteonectin. The strategy is straightforward, results in large amounts of material, and is universally applicable for the generation of protein domain chimeras. In some cases 10% dimethyl sulfoxide aided the amplification. Four base CCGC “clamp” sequences adjacent to BamHI restriction sites at the ends of the PCR products were used to enhance the ligation of products. A hybrid inverse complement oligonucleotide primer composed of sequences containing 20 nucleotides of protein S and 16 nucleotides of osteonectin was used in the first round of PCR. An additional osteonectin sequence was added to the initial amplified product by performing PCR using a second “boot-strap” primer containing 18 nucleotides of osteonectin. Primers used to amplify osteonectin encompassed the 146-amino-acid NH 2-terminal half of osteonectin. The double-stranded first-round fragments of protein S-osteonectin and osteonectin were subsequently mixed together and one elongation cycle of PCR was performed. Annealing occurred as the result of the 34-base-pair overlap region composed of osteonectin sequence. Taq polymerase was used for elongation with subsequent recombinant DNA synthesis. After elongation, external primers were added to amplify the protein S-osteonectin gene construct. The protocol we have developed allows noncoding and coding segments of DNA to be linked, GC-rich areas of DNA to be amplified, hybridization temperatures to be increased, annealing times to be reduced, and PCR of products to be subcloned.

The murine gene encoding secreted phosphoprotein 1 (osteopontin): promoter structure, activity, and induction in vivo by estrogen and progesterone

Secreted phosphoprotein 1 (Spp-1) is a 41.5-kDa bone sialoprotein presumed to be important in the development and functioning of a number of mammalian organs and possibly also in the progression of malignancies. We report here the isolation of a phage λ genomic clone of the murine Spp-1 gene containing the promoter and first six exons (4.6 kb of the 5.7-kb gene). We have found another exon located 5′ to the ‘exon 1’ reported by Miyazaki et al. [J. Biol. Chem. 265 (1990) 14432–14438]. The DNA upstream from this 5′ exon functions as a promoter in epidermal fibroblast and osteoblast-like cells, as demonstrated by transient transfection assays, S 1 mapping of the transcription start point, and sequence analysis revealing TATA-like (TTTAAA) and CAAT (its inverse complement) boxes. A small region of the promoter (nt −253 to + 79) was able to direct high-level expression of a fused cat reporter gene in JB6 mouse epidermal cells. The transient transfection assays indicated the presence of a positive transcription element between nt −543 and −253 and a negative transcription element between nt −777 and −543. Addition of the tumor promoter, 12- O-tetradecanoylphorbol-13-acetate (TPA), resulted in a 1.5-3-fold induction of transcription, depending on the promoter construct and the TPA concentration. The Spp-1 mRNA was localized in several tissues, consistent with previous reports, and to novel sites in ovary, and in the skin and ventral fatty tissue of pregnant and lactating mice. The induction of Spp-1 mRNA was partially mimicked by painting β-estradiol or progesterone on the skin of nonpregnant females.

Sequence and uptake specificity of cloned sonicated fragments of Haemophilus influenzae DNA

Our data support the conclusion that all tight binding of DNA by competent cells of Haemophilus influenzae is due to 9 base pairs (bp) of the 11-bp motif 5'-AAGTGCGGTCA or its inverse complement, TGACCGCACTT. Sonicated chromosomal DNA of H. influenzae was absorbed and extracted from competent cells to prepare a subpopulation of uptake fragments enriched for single uptake (binding) sequences. This DNA was inserted into the plasmid pUC18 and cloned into Escherichia coli. Whole sonicated chromosomal DNA was also cloned in pUC18 as a population of control fragments. Seventy-one plasmids were labeled with 3H and tested for DNA binding, and the inserts of 28 of them were sequenced. The control plasmid pUC18 was absorbed to competent H. influenzae cells at low levels of 5 to 10% of DNA added. The plasmids containing uptake inserts were mostly absorbed at levels of 70 to 80%, but a significant number contained inserts with an intermediate level of binding, 20 to 30%. The inserts of 15 plasmids were excised and tested for DNA uptake to demonstrate good agreement between uptake of these plasmids and their insert fragments. Sequencing of inserts revealed that the presence of 9 bp of the 11-bp motif was associated with tight binding. Some inserts with intermediate levels of binding had no significant similarities to the 9-bp sequence. The 9-bp sequence appeared to account for most of the binding to competent cells, but appreciable binding occurred with fragments without 9-bp homology. About one-third of the 9-bp uptake sequences were found as inverted repeats that could form strong stem-loop structures. It has been suggested by Goodman and Scocca (Proc. Nal. Acad. Sci. USA 85:6982-6986, 1988) that in Neisseria gonorrhoeae, uptake sequences occur as palindromes and act as transcription terminators. Although consistent, the data are insufficient to make this conclusion for uptake sequence palindromes in H. influenzae.

Complementary hydropathy and the evolution of interacting polypeptides

It has been shown that codons coding for strongly hydrophilic amino acids are complemented by codons that code for strongly hydrophobic ones, leading to a hypothesis stating that peptides thus encoded should interact. Though the principle has been validated in a number of experimental models, its general applicability has been questioned. I have discussed this principle, showing that the correlation between coding and noncoding strand amino acids was maintained, indeed slightly improved, when weighted averages based on codon usage tables were used to determine noncoding strand amino acid hydropathies. The coding capacity of the noncoding strand and its content of open reading frames were also discussed. Another point of contention that was afforded further clarification is the chemical plausibility of interactions between hydrophobic and hydrophilic amino acids implicit in this concept. The extension of complementary domains was also dealt with. Finally, I have discussed what I called the evolutionary drift of primary structure, and I showed as an example that though nucleotide sequences coding for the substance K receptor bear little resemblance to the inverse complement of that which codes for the SK peptide, a peptide spanning residues 130–139 is hydropathically very similar to that predicted from such an inverse complement.

Characterization of chicken octamer-binding proteins demonstrates that POU domain-containing homeobox transcription factors have been highly conserved during vertebrate evolution.

The DNA sequence motif ATTTGCAT (octamer) or its inverse complement has been identified as an evolutionarily conserved element in the promoter region of immunoglobulin genes. Two major DNA-binding proteins that bind in a sequence-specific manner to the octamer DNA sequence have been identified in mammalian species--a ubiquitously expressed protein (Oct-1) and a lymphoid-specific protein (Oct-2). During characterization of the promoter region of the chicken immunoglobulin light chain gene, we identified two homologous octamer-binding proteins in chicken B cells. When the cloning of the human gene for Oct-2 revealed it to be a member of a distinct family of homeobox genes, we sought to determine if the human Oct-2 cDNA could be used to identify homologous chicken homeobox genes. Using a human Oct-2 homeobox-specific DNA probe, we were able to identify 6-10 homeobox-containing genes in the chicken genome, demonstrating that the Oct-2-related subfamily of homeobox genes exists in avian species. Low-stringency screening of a chicken embryonic cDNA library allowed us to clone one of these genes. DNA sequence analysis revealed it to be the chicken homologue of the human Oct-1 gene. The predicted protein sequence of the chicken Oct-1 gene demonstrated that the gene for Oct-1 has been highly conserved during vertebrate evolution with an overall 96% amino acid sequence identity between the chicken and human proteins. The previously described POU domain (termed POU for its presence in the Pit-1, Oct-1/Oct-2, and Unc-86 genes) and homeobox domain are 100% conserved between the two protein products. Together, our data show that the POU-containing subfamily of homeobox genes have been highly conserved during vertebrate evolution, apparently as a result of selection for their DNA-binding and transcriptional regulatory properties.

Is there a relationship between DNA sequences encoding peptide ligands and their receptors?

It has been suggested that the coding for a ligand and its receptor may have originated in inverse complementary strands of the same DNA. This would imply a deficiency of stop codons in the complementary strand of the ligand message sequence. We have sought evidence of such deficiencies by an analysis of the usage of selected codons in 23 human neuropeptide and hormone mRNA sequences. We have also searched directly for similarities between substance K or substance P and the substance K receptor. Although bovine proopiomelanocortin has an open reading frame for the full extent of the inverse complement of the coding region, this seems to be a unique case. The data as a whole do not support the hypothesis.

An octamer oligonucleotide upstream of a TATA motif is sufficient for lymphoid-specific promoter activity.

The octamer sequence ATGCAAAT or its inverse complement ATTTGCAT is well-conserved in all immunoglobulin gene promoters and has been implicated in promoter function by deletion analysis. Although immunoglobulin promoters are tissue-specific, the octamer is also a functional element in non-tissue-specific upstream regions--like those controlling U1 and U2 small nuclear RNA and histone H2B genes--where it is associated with additional canonical elements. Specific interactions occur between the octamer motif and both lymphoid-specific and ubiquitous proteins. By using a synthetic octamer oligonucleotide inserted upstream of the beta-globin TATA box we show here that the octamer element by itself is sufficient for directing lymphocyte-specific RNA synthesis when within 70 base pairs of the start site of transcription. We also demonstrate that mutations in any position of the conserved motif interfere with this function.

Purification of an octamer sequence (ATGCAAAT)-binding protein from human B cells.

The highly conserved octamer sequence ATGCAAAT or its inverse complement found in all human and murine immunoglobulin gene promoters has been demonstrated to be necessary in the lymphoid-specific transcription by deletion analysis. Trans-acting factors that interact with the octamer motif are thought to be involved in this tissue-specific expression. Using a gel mobility shift assay, we have identified both lymphoid-specific and ubiquitous nuclear factors that interact with a human gamma 1 heavy chain gene promoter region containing the octamer motif, consistent with the results obtained with murine heavy or light chain promoter regions. We have purified an octamer binding protein from human B cells by sequence-specific DNA affinity chromatography. Renaturation of gel-purified protein allowed the identification of a polypeptide with a molecular weight of 74 kilodaltons (kD) that is capable of recognizing and binding to the octamer motif. This 74 kD protein seems to be also present in T-cells and non-lymphoid cells. The possible function of the factor is discussed.

Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. II. Isolation of the gene and characterization of the 5' flanking region.

Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and microsomal HMG-CoA reductase are sequential enzymes in the cholesterol biosynthetic pathway; both are negatively regulated by cholesterol. In this paper, we report the isolation of overlapping bacteriophage lambda clones that encompass the gene for hamster cytoplasmic HMG-CoA synthase. The gene spans 20 kilobases and contains 11 exons and 10 introns. Under conditions of high-level expression in cultured hamster cells and hamster liver, approximately 50% of the mRNAs contain two introns in the region of the gene corresponding to the 5' untranslated region. The remaining 50% contain only one intron as a result of the direct splicing of exon 1 to exon 3. The optional exon (exon 2) in the 5' untranslated region contains a 26-nucleotide sequence that is homologous to the 5' untranslated region of the mRNA for HMG-CoA reductase. Approximately 350 base pairs upstream of the transcription initiation site, the HMG-CoA synthase gene contains a sequence that is strongly homologous to the 72-base pair enhancer region of the SV40 virus. Approximately 125 nucleotides downstream of this region, the sequence CCGCCC, or its inverse complement GGGCGG, is repeated four times. Multiple copies of this sequence are present in the SV40 promoter and in the 5' flanking region of HMG-CoA reductase and several other cellular housekeeping genes. The availability of cloned genes for two consecutive negatively regulated enzymes of the cholesterol synthetic pathway should allow elucidation of the mechanism for this coordinate expression.