CYB Gene Research Articles

Mitochondrial Pathogenic Mutations and Expression Pattern of Oxidative Phosphorylation Genes in COVID-19 Patients.

Mitochondrial missense mutations and pathogenic variants have been implicated in the pathogenesis of COVID-19. This study evaluated the role of mitochondrial DNA (mtDNA) mutations and changes in gene expression in the progression of COVID-19 and their correlation with clinical characteristics. Next-generation sequencing with high throughput was used to identify mtDNA mutations in 30 COVID-19 patients compared to 20 healthy controls. The potential impact of identified mutations on protein structure and stability was predicted using bioinformatic tools. Quantitative real-time polymerase chain reaction was employed to assess the expression levels of mtDNA-encoded genes involved in oxidative phosphorylation in COVID-19 patients and healthy controls. Correlations between gene expression levels, clinical parameters, including leukocyte, lymphocyte, neutrophil, and platelet count, as well as creatinine, alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) levels, and disease severity were analyzed. We found 8 different mtDNA mutations in ND1, ND5, CO3, ATP6, and CYB genes, which were predicted to alter amino acids and decrease protein stability. Two missense unique mutations, C9555T in CO3 and A12418T in ND5 were identified and correlated with Complexes I and IV, respectively. This downregulation was correlated with age, elevated levels of leukocytes, lymphocytes, neutrophils, platelets, creatinine, ALT, AST, and BUN, as well as disease severity. These findings suggest that mtDNA mutations and altered expression of oxidative phosphorylation genes contribute to mitochondrial dysfunction in COVID-19. Targeting mitochondrial dysfunction may represent a promising therapeutic strategy for COVID-19 treatment.

Prognostic genome and transcriptome signatures in colorectal cancers

Colorectal cancer is caused by a sequence of somatic genomic alterations affecting driver genes in core cancer pathways1. Here, to understand the functional and prognostic impact of cancer-causing somatic mutations, we analysed the whole genomes and transcriptomes of 1,063 primary colorectal cancers in a population-based cohort with long-term follow-up. From the 96 mutated driver genes, 9 were not previously implicated in colorectal cancer and 24 had not been linked to any cancer. Two distinct patterns of pathway co-mutations were observed, timing analyses identified nine early and three late driver gene mutations, and several signatures of colorectal-cancer-specific mutational processes were identified. Mutations in WNT, EGFR and TGFβ pathway genes, the mitochondrial CYB gene and 3 regulatory elements along with 21 copy-number variations and the COSMIC SBS44 signature correlated with survival. Gene expression classification yielded five prognostic subtypes with distinct molecular features, in part explained by underlying genomic alterations. Microsatellite-instable tumours divided into two classes with different levels of hypoxia and infiltration of immune and stromal cells. To our knowledge, this study constitutes the largest integrated genome and transcriptome analysis of colorectal cancer, and interlinks mutations, gene expression and patient outcomes. The identification of prognostic mutations and expression subtypes can guide future efforts to individualize colorectal cancer therapy.

Higher-order co-mutation interactions in mitochondrial genomes

Pair-wise co-mutation networks of the mitochondrial genome have already provided ample evidences about the roles of genetic interactions in the manifestation of phenotype under altered environmental conditions. Here, we present a method to construct and analyze higher-order interactions, namely, three-uniform hypergraphs of the mitochondrial genome for different altitude populations to decipher the role of co-mutating variable sites beyond pair-wise interactions. We considered the human mitochondrial DNA residing at different altitudes with respect to Tibet in Asia as a case study. We found that in 50% of the gene triangles, two positions were occupied by coding genes, which suggested that coding genes are dominantly involved in forming the hyperedges. Based on weights of the gene triangles, we identified altitude-specific genes such as, in low-altitude ATP6 and ND genes, in mid-altitude CO1 and ND genes, and in high-altitude ATP6, CO1, CYB and ND genes. This framework of three-uniform hypergraph serves an avenue for future investigation of nuclear genomes in context of phenotypic association and genetic disorders beyond the pair-wise interactions.

Clinical and genetic analysis of essential hypertension with CYB gene m.15024G>A mutation.

To explore the role of mitochondrial CYB 15024G>A mutation in the development of essential hypertension. Mitochondrial genome sequences of hypertensive patients were obtained from previous studies. Clinical and genetic data of a hypertensive patient with mitochondrial CYB 15024G>A mutation and its pedigree were analyzed. Lymphocytes derived from patient and family members were transformed into immortalized lymphoblastoid cell lines, and the levels of adenosine triphosphate (ATP), mitochondrial membrane potential and intracellular reactive oxygen species (ROS) were detected. The penetrance of this essential hypertension family was 42.9%, and the age of onset was 46-68 years old. Mitochondrial genome sequencing results showed that all maternal members carried a highly conserved mitochondrial CYB 15024G>A mutation. This mutation could affect the free energy of mitochondrial CYB for secondary and tertiary structure and protein folding, thereby changing its structural stability and the structure of the electron transfer function area around the mutation site. Compared with the control, the cell line carrying the mitochondrial CYB 15024G>A mutation showed significantly decreased levels of mitochondrial CYB, ATP and mitochondrial membrane potential, and increased levels of ROS (P<0.01). Mitochondrial CYB 15024G>A mutation may affect the structure of respiratory chain subunits and mitochondrial function, leading to cell dysfunction, which suggests that the mutation may play a synergistic role in essential hypertension.

The Italian wall lizard, Podarcis siculus campestris, unexpected presence on Gorgona Island (Tuscan Archipelago)

We here report the unexpected presence of the Italian wall lizard (Podarcis siculus campestris) on Gorgona Island, in the Parco Nazionale Arcipelago Toscano (Tuscan Archipelago, Tyrrhenian Sea, Tuscany, Central Italy). Field observations were carried out in 2020 confirming its presence on the island, where it had never been reported before. We recorded 37 GPS points of the species in three major areas of Gorgona (with 50 lizard records) and about 180 visual counts regarding all age classes (newborns, juveniles and adults). The species was found in the urban area (site of state prison) and in two grassy and bushed areas, around and along olive tree plantations. Seven individuals were captured and their tails were used to assess the sequence variation of the mitochondrial CYB gene. Biometrical parameters were also evaluated for six of these individuals. We detected three distinct CYB haplotypes that were compared to Podarcis siculus CYB sequences available in public databases. They resulted identical or phylogenetically closest to those found in mainland Tuscany. One haplotype, found in three specimens, was identical to one previously detected at Orti Bottagone (WWF Oasis in Piombino), while the other two haplotypes were most similar to haplotypes reported in the Giannella peninsula and Pisa, respectively.

Role of mitochondrial genetic interactions in determining adaptation to high altitude human population

Physiological and haplogroup studies performed to understand high-altitude adaptation in humans are limited to individual genes and polymorphic sites. Due to stochastic evolutionary forces, the frequency of a polymorphism is affected by changes in the frequency of a near-by polymorphism on the same DNA sample making them connected in terms of evolution. Here, first, we provide a method to model these mitochondrial polymorphisms as “co-mutation networks” for three high-altitude populations, Tibetan, Ethiopian and Andean. Then, by transforming these co-mutation networks into weighted and undirected gene–gene interaction (GGI) networks, we were able to identify functionally enriched genetic interactions of CYB and CO3 genes in Tibetan and Andean populations, while NADH dehydrogenase genes in the Ethiopian population playing a significant role in high altitude adaptation. These co-mutation based genetic networks provide insights into the role of different set of genes in high-altitude adaptation in human sub-populations.

PCR-RFLP Design for Authentication of Red Snapper Species Based on CYB Gene

The PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique is a species identification method that can facilitate food inspection agencies to overcome mislabelling. In addition, this technique is simple, fast, powerful, and cheap compared to DNA barcodes. This study aimed to accommodate the problem in determining restriction endonuclease enzymes for the digestion of PCR end products through the design of snapper species authentication using the CYB gene in silico. Differentiation of CYB gene sequences with DNA barcoding showed that all species could be differentiated with the highest similarity level in Red Fish (Sebastes) species by 98.9% and snapper species between L. malabaricus and L. erythropterus by 99.8%. Enzyme tracing based on sequences variation of snapper species with substitution species found three potential enzymes from the CYB gene sequence, namely, Accl, Fnu4HI, and Tsp45I. Where all these enzymes can discriminate red snapper species among other.Key words: PCR-RFLP; CYB gene; Red snapperAbstrakTeknik PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) merupakan metode identifikasi spesies yang dapat memfasilitasi lembaga inspeksi makanan untuk mengatasi mislabelling. Selain itu, teknik ini sederhana, cepat, kuat, dan murah dibandingkan dengan barcode DNA. Penelitian ini bertujuan untuk mengakomodasi masalah dalam menentukan enzim restriksi endonuklease untuk digesti produk akhir PCR lewat perancangan autentikasi spesies ikan kakap menggunakan gen CYB secara in silico. Diferensiasi sekuens gen CYB dengan DNA barcoding menunjukkan semua spesies dapat dibedakan dengan tingkat kesamaan tertinggi pada spesies Red Fish (Sebastes) sebesar 98,9% dan spesies kakap antara L. malabaricus dan L. erythropterus sebesar 99,8%. Penelusuran enzim berdasarkan variasi sekuens spesies kakap dengan spesies substitusi ditemukan sebanyak tiga enzim yang berpotensi yaitu, Accl, Fnu4HI, dan Tsp45I. Semua enzim tersebut dapat memdiskriminasikan spesies kakap merah dari spesies lainnya.Kata kunci: PCR-RFLP; gen CYB; ikan kakap merah

Analysis of compositional properties and codon usage bias of mitochondrial CYB gene in anura, urodela and gymnophiona.

We delineated the pattern of synonymous codon usage bias (CUB) and its determinants in mitochondrial CYB gene of respiratory chain across different amphibian groups namely orders anura, urodela and gymnophiona. We observed that CUB was low in CYB gene of amphibia. The gymnophionans had comparatively high bias followed by urodeles and anurans. The codons namely TCA, CCA, CAA, CGA, TGA, AAA and ACA were over-represented in all three orders. The codons such as GCC and TCC were over-represented in anura whereas in urodela, the over-represented codons were TTA, CTA, ATA, GTA, GAA, GGA and GCA. In gymnophiona, GCC, TTA, CTA, ATA, GTA, GAA and GGA codons were over-represented. The regression analysis between effective number of codons (ENC) and nucleobase at the 3rd position revealed that nucleobase A and C influenced CUB positively in order anura, while in urodela and gymnophiona, nucleobase A and T influenced the CUB positively. Mutation pressure and natural selection mutually illustrate the CUB of CYB gene (complex III gene) of amphibia as elucidated by correlation analysis between 3rd nucleotide in a codon and overall nucleotide content of the gene. However, neutrality plot showed that natural selection was the dominant evolutionary factor of CUB.

Heteroplasmic Mutations and Polymorphisms in the Cyb Gene of Mitochondrial DNA in Canine Mast Cell Tumours.

Identification of mutations and polymorphisms in the cytochrome b gene (Cyb) of mitochondrial DNA (mtDNA) in canine mast cell tumours and determinatiion of their association with the process of neoplastic transformation. The samples comprised tumour tissues and blood obtained from 34 dogs of various breeds. Mutations and polymorphisms in the Cyb gene were detected using amplification and sequencing methods. Heteroplasmic mutations were detected at seven positions of mtDNA in 86% of the individuals. Blood and tumour heteroplasmy were recorded at five nucleotide positions of the Cyb gene, whereas tumour heteroplasmy was detected at two positions. Polymorphisms were detected at 14 Cyb gene positions in in the blood of 91% of dogs with mast cell tumours. The presence of numerous mutations and polymorphisms of Cyb in the blood and tumour tissues and the high frequency of heteroplasmy indicate their involvement in the process of neoplastic transformation in dogs.

A cross-talk on compositional dynamics and codon usage patterns of mitochondrial CYB gene in Echinodermata

Codon usage bias (CUB) refers to a phenomenon in which some synonymous codons are used in mature mRNA at a higher frequency than other members codifying the same amino acid. CUB is mainly determined by mutation pressure and natural selection. We used bioinformatic tools to analyze the protein coding sequences of mitochondrial CYB gene in different classes of Echinodermata to understand the patterns of codon usage. The ENC values of CYB gene in five different classes of Echinodermata were 41.64, 30.33, 43.63, 41.11, and 41.33, which suggested that the CUB of this gene was low. The relative synonymous codon usage (RSCU) values showed that the patterns of over-represented and under-represented codons were different among different classes. Correspondence analysis indicated that the plots of CYB gene were different across classes, suggesting that the pattern of codon usage was also different among five classes under study. Highly significant correlation (p < .01) between overall nucleotide composition and its 3rd codon position indicated that both mutational pressure and natural selection had an influence on the codon usage bias of CYB gene. Furthermore, PR-2 bias plot analysis showed that both mutation pressure and natural selection might have affected the pattern of codon usage in CYB gene of Echinodermata.

Association study of mitochondrial genetic polymorphisms in asthmatic children

It has been suggested that mitochondrial dysfunction plays a role in the pathogenesis of asthma. To test whether mitochondrial variants influence the risk of asthma, we analyzed 16,158 mtSNPs in a sample of 372 asthmatic children and 395 healthy children using the DNA pooling technique and genome wide association analysis. Stratified analysis by sex was performed to explain the differences observed between sexes in the etiology of asthma. Different variants were detected to be significant in the sample of girls and boys with the smallest adjusted p values being 1.4×10−09 (mt5295) and 3.6×10−12 (mt16158), respectively. Most of the significant locations found in boys are within the CYB gene and the non-coding region. For girls, most of the significant mtSNPs lie within NADH-dehydrogenase-subunits. The variants reported here have not previously been described in connection with asthma. Although further studies in other cohorts are needed to confirm these findings our study highlights the importance of the mitochondria among the factors that contribute to the risk of asthma.

Genetic characterization of banteng (Bos javanicus) in Lam Pao Wildlife Conservation Development and Promotion Station, Kalasin Province

We analyzed the mitochondrial cytochrome b sequences of bantengs (Bos javanicus) domesticated in Lam Pao Wildlife Conservation Development and Promotion Station, Kalasin Province, which were donated by the Indonesian government. A total of 373 nucleotide and 117 amino acid positions of the Cyb gene were sequenced and compared among the banteng isolates as well as other species of Bovidae. The bantengs domesticated in Kalasin showed 100% identity with the Indonesian bantengs and were more closely clustered with the gaur, Bos gaurus and Bos frontalis instead of a clade of Thai and Cambodian bantengs. Thus, the Kalasin banteng represent a pure strain of B. javanicus, Indonesian banteng.

Extensive screening system using suspension array technology to detect mitochondrial DNA point mutations

We established an extensive and rapid system using suspension array to detect 61 representative mitochondrial DNA (mtDNA) heteroplasmic or homoplasmic point mutations (29 for Series A and 32 for Series B) in 22 genes: 1 each in MT-RNR1, -TV, -ND1, -TQ, -TW, -TC, and -TH genes; 2 each in MT-TN, -TG, -ND4, -TL2, -TE, and -CYB genes; 3 each in MT-ATP6, -ND3, and -ND5 genes; 4 each in MT-CO1 and -TK genes; 5 each in MT-TI, -TS1, and -ND6 genes; and 10 in the MT-TL1 gene. We carefully selected 5′-biotinylated primers and pooled primers for use in two sets of multiplex-PCR amplifications. To detect both mutant and wild-type mtDNA, even when polymorphisms were present near the target mutation sites, we designed specific oligonucleotide probes. By using the mtDNA point mutation detection system of Series A (29 mutations) and Series B (32 mutations), we screened a total of 3103 mutant sites in 107 DNA samples for Series A and 13,101 mutant sites in 397 DNA samples for Series B. We succeeded in determining 99.4% (Series A) and 99.6% (Series B) of the targeted mutant sites by use of the system. The 22 samples with the m.3243A>G heteroplasmic mutation revealed positive signals with both mutant- and wild-type-specific probes in this detection system with a detection limit of approximately 2%. This genetic screening platform is useful to reach a definitive diagnosis for mitochondrial diseases.

C. elegans mitotic cyclins have distinct as well as overlapping functions in chromosome segregation

Mitotic cyclins in association with the Cdk1 protein kinase regulate progression through mitosis in all eukaryotes. Here, we address to what extent mitotic cyclins in the nematode Caenorhabditis elegans provide overlapping functions or distinct biological activities. C. elegans expresses a single A-type cyclin (CYA-1), three typical B-type cyclins (CYB-1, CYB-2.1 and CYB-2.2), and one B3-subfamily member (CYB-3). While we observed clear redundancies between the cyb genes, cyb-1 and cyb-3 also contribute specific essential functions in meiosis and mitosis. CYB-1 and CYB-3 show similar temporal and spatial expression, both cyclins localize prominently to the nucleus, and both associate with CDK-1 and display histone H1 kinase activity in vitro. We demonstrate that inhibition of cyb-1 by RNAi interferes with chromosome congression and causes aneuploidy. In contrast, cyb-3(RNAi) embryos fail to initiate sister chromatid separation. Inhibition of both cyclins simultaneously results in a much earlier and more dramatic arrest. However, only the combination of cyb-1, cyb-3 and cyb-2.1/cyb-2.2 RNAi fully resembles cdk-1 inhibition. This combination of redundant and specific phenotypes supports that in vivo phosphorylation of certain Cdk targets can be achieved by multiple Cdk1/cyclin complexes, while phosphorylation of other targets requires a unique Cdk1/cyclin combination.

The mitochondrial genome can be altered or lost without lethal effect in the petite-negative yeast Debaryomyces (Schwanniomyces) occidentalis.

The nature of mutations affecting several cytochrome-deficient mutants of Debaryomyces (Schwanniomyces) occidentalis has been characterized. The DR12 mutant, which is deficient in cytochrome b, and the B10Mn mutant, which is deficient in cytochromes b and a, a3, are deleted in the mitochondrial CYB and COX1 genes respectively. The B10 strain, which is partially deficient in cytochrome b, has no detectable change in its mitochondrial DNA and possibly carries nuclear lesion(s). These three mutants, unlike the rho(-) and rho degrees "petite" mutants of Saccharomyces cerevisiae, can still grow on non-fermentable substrates, due to the development of a salicylhydroxamic acid (SHAM)-sensitive alternative pathway linked to phosphorylation at site 1. A gly(-) mutant lacking mtDNA and respiratory capacity has been isolated. For the first time, it is demonstrated that mtDNA can be altered or even lost without lethal consequence in D. occidentalis, although this yeast was classified as a petite-negative species.

Inhibition of mitochondrial and plastid activity of Plasmodium falciparum by minocycline

We previously reported the superior effect of minocycline against drug-resistant Plasmodium falciparum in vitro. Here, we report that RT-PCR for falciparum parasites treated with minocycline revealed reduced levels of RNA transcripts of the mitochondrion-encoded genes such as the COI and Cyb genes, as well as the plastid-encoded RNA polymerase subunit (rpoB/C) gene. However, we detected no apparent effects of the antibiotic on the transcription of merozoite surface antigen and small subunit rRNA genes encoded by the nucleus. In addition, treatment with chloroquine and pyrimethamine showed no substantial reduction of any RT-PCR products. These findings suggest that tetracycline antibiotics selectively inhibit both mitochondrial and plastid activity.

Complete nucleotide sequence of the 6 kb element and conserved cytochrome b gene sequences among Indian isolates of Plasmodiumfalciparum

The malaria parasite contains a nuclear genome with 14 chromosomes and two extrachromosomal DNA molecules of 6 kb and 35 kb in size. The smallest genome, known as the 6 kb element or mitochondrial DNA, has been sequenced from several Plasmodium falciparum isolates because this is a potential drug target. Here we describe the complete nucleotide sequence of this element from an Indian isolate of P. falciparum. It is 5967 bp in size and shows 99.6% homology with the 6 kb element of other isolates. The element contains three open reading frames for mitochondrial proteins-cytochrome oxidase subunit I (CoI), subunit III (CoIII) and cytochrome b (Cyb) which were found to be expressed during blood stages of the parasite. We have also sequenced the entire cyb gene from several Indian isolates of P. falciparum. The rate of mutation in this gene was very low since 12 of 14 isolates showed the identical sequence. Only one isolate showed a maximum change in five amino acids whereas the other isolate showed only one amino acid change. However, none of the Indian isolates showed any change in those amino acids of cyb which are associated with resistance to various drugs as these drugs are not yet commonly used in India.

Antimycin A resistance in a mutant Leishmania tarentolae strain is correlated to a point mutation in the mitochondrial apocytochrome b gene.

In this paper we report the first case of antimycin A resistance in a protozoan parasite that is attributable to a mutation in the mitochondrial apocytochrome b (CYb) gene. We selected for, and isolated, a mutant Leishmania tarentolae strain that is resistant to antimycin A. This resistance was evident at the levels of the in vitro growth and enzymatic activity of the cytochrome bc1 complex. Molecular characterisation of the mutant revealed a Ser35Ile mutation in the expected region of the CYb gene. In kinetoplastids, CYb and other structural genes of the mitochondrial genome are located on the maxicircle component of the mitochondrial DNA, which is present in 20-50 copies. Primer-extension analysis confirmed the presence of the mutation at the mRNA level. The phenotypic manifestation of the mutation implies that the CYb mRNA is edited and translated within the mitochondrion. Thus, this finding provides direct evidence that edited RNAs are translated in kinetoplastid mitochondria. Furthermore, a defined mutation conferring drug resistance to a mitochondrial gene product can be exploited for the development of mitochondrial transfection systems for trypanosomatids.

Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation

The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 x 10(-7)) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 x 10(-7)). No colonies were seen on control plates (frequency < 0.96 x 10(-9)). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.

Characterization of sequence changes in kinetoplast DNA maxicircles of drug-resistant Leishmania

We have compared kinetoplast DNA maxicircles of tunicamycin- and arsenite-resistant variants of repeatedly cloned Leishmania mexicana amazonensis showing DNA amplification with wild-type and arsenite-resistant variants of the same lineage that do not show DNA amplification. DNA restriction patterns and the degree of cross-hybridization between maxicircle DNA fragments of parasites displaying DNA amplification and those of parasites without amplification were examined. In addition, the nucleotide sequence of the cytochrome b (Cyb) gene from the coding region was compared between these two groups of parasites. Extensive changes were found in the nucleotide sequences and the amino acid sequences of the cytochrome gene of the maxicircles of variants with DNA amplification. The Cyb genes from both groups had much shorter open reading frames than the same gene from Leishmania tarentolae and Trypanosoma brucei. The simultaneous changes in maxicircles and minicircles of these variants suggest that they may confer the advantage of maintaining viable mitochondrial function under selective pressure.