Nonsynonymous Nucleotide Substitutions Research Articles

The Implication of Codon Usage Design and Expression Level in Determining the Nature of Selection and Functionality Amongst the Amino Acid Biosynthetic Pathway Coding Sequences of Arthrobacter sp. FB24

Amino acid biosynthesis is an immensely important life process requiring the participation of nearly hundred gene products, differing in terms of codon usage pattern, expressivity and selection pressure. In this study an attempt was made to categorize the gene products according to their expression level, nature of selection and functionality analyzing the amino acid biosynthetic pathway genes of the soil bacterium Arthrobacter, known for its varied metabolic activities and a key agent for industrial production of amino acids. The relationship between the different parameters like expression level, codon usage and selection pressure was calculated to find out the correlation of biasness, expression level, nature of selection and functionality amongst the genes involved in amino acid biosynthetic pathways. We found that genes coding for enzymes and proteins with large number of functions have higher codon usage bias and are subjected mostly to purifying selection. Gene sequences of proteins and enzymes with low functionality showed less codon usage bias and are under positive selection. The multifunctional genes mostly show high expression level and it is highly correlated with codon usage bias. The results of this study demonstrate that functionality and nature of selective forces acting on the coding sequences can be determined by studying the correlation between the different parameters like effective number of codon, codon adaptation index and nucleotide substitution ratios and careful study of all these parameters is a pretty good indicator for determining the functionality of the genes and this can be extended towards the genes of unknown function(s). Keywords: Amino acid biosynthetic pathways, Arthrobacter, CAI, Nc, Ka/Ks, synonymous and non-synonymous nucleotide substitution rates, positive selection, purifying selection.

Detection and genetic analysis of the enteroaggregative Escherichia coli heat-stable enterotoxin (EAST1) gene in clinical isolates of enteropathogenic Escherichia coli (EPEC) strains.

BackgroundThe enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) encoded by astA gene has been found in enteropathogenic E. coli (EPEC) strains. However, it is not sufficient to simply probe strains with an astA gene probe due to the existence of astA mutants (type 1 and type 2 SHEAST) and EAST1 variants (EAST1 v1-4). In this study, 222 EPEC (70 typical and 152 atypical) isolates were tested for the presence of the astA gene sequence by PCR and sequencing.ResultsThe astA gene was amplified from 54 strains, 11 typical and 43 atypical. Sequence analysis of the PCR products showed that 25 strains, 7 typical and 18 atypical, had an intact astA gene. A subgroup of 7 atypical strains had a variant type of the astA gene sequence, with four non-synonymous nucleotide substitutions. The remaining 22 strains had mutated astA gene with nucleotide deletions or substitutions in the first 8 codons. The RT-PCR results showed that the astA gene was transcribed only by the strains carrying either the intact or the variant type of the astA gene sequence. Southern blot analysis indicated that astA is located in EAF plasmid in typical strains, and in plasmids of similar size in atypical strains. Strains carrying intact astA genes were more frequently found in diarrheic children than in non-diarrheic children (p < 0.05).ConclusionIn conclusion, our data suggest that the presence of an intact astA gene may represent an additional virulence determinant in both EPEC groups.

In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment.

Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK, which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrB(L82R) in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrB(L82R) mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

1013-lbp: identification of 2127 new HLA alleles in potential donors registered with DKMS donor centers in Germany, the United States and Poland

We describe 2127 new HLA alleles: 598 HLA-A, 755 HLA-B and 774 HLA-C alleles, accounting for 28.9 % of the currently known HLA class I alleles. Buccal swabs or blood samples of newly recruited donors from three national DKMS donor centers were typed for HLA using sequencing-based typing (SBT) at the ASHI-accredited laboratory of HistoGenetics (Ossining, NY). New HLA class I alleles were identified after determination of DNA sequences of HLA exons 2 and 3. Comparison of the new allele sequences and their most homologous counterpart lead to detection and description of nucleotide variations in DNA sequences. Self-assessed parentage of individuals carrying new HLA class I alleles were examined. Of the 2127 new HLA class I alleles described, 1336 (62.8%) were observed in donors from Germany, 422 (19.8%) in US donors, 213 (10.0%) in donors from Poland, and 156 (7.3%) new alleles were found in donors from at least two countries. We identified new alleles in 57 allele groups, including 192 C ∗ 07, 144 A ∗ 02 and 112 C ∗ 03 alleles, The majority (93.8%) of the new alleles corresponded to single nucleotide variations. 67.0% of the new alleles comprised non-synonymous nucleotide substitutions and 30.0% silent mutations. The remaining 3.0% showed nonsense mutations identified in null alleles. New alleles were found disproportionally often in minority donors. Further, 527 new alleles (24.8%) were identified in multiple individuals. We described 2127 new HLA class I alleles that have been identified in upfront HLA typing of newly registered DKMS stem cell donors. The occurrence of many new alleles in multiple individuals who originated partly from different populations indicates that newly identified alleles are not necessarily rare and thus may be of relevance for actual stem cell donor searches.

Detecting mitochondrial signatures of selection in wild Tibetan pigs and domesticated pigs

Selection in genomic regions is prevalent in mammals; however, the effects of selection on the mitogenome are not clearly understood. We determined the complete mitochondrial DNA (mtDNA) sequences from six wild Tibetan pigs from the Tibetan plateau and four domestic pig breeds from the lowland of neighboring southwest China. Nucleotide diversity analysis using the sliding window method showed that the nucleotide diversity of wild Tibetan pigs in most regions of the mitogenome was higher than that of domestic pigs. The 12 s ribosomal RNA showed relatively lower nucleotide diversity in Tibetan pigs, suggesting purifying selection of these genes during high-altitude adaptation. More non-synonymous nucleotide substitutions in the ATP6 were found in wild Tibetan pigs, indicating adaptive selection in Tibetan pigs. The results suggested distinct impacts of natural selection and artificial selection upon the mitogenome, especially the mitochondrial signatures of adaptive evolution in wild Tibetan pigs under natural selection.

Phylogeny and patterns of diversity of goat mtDNA haplogroup A revealed by resequencing complete mitogenomes.

We sequenced to near completion the entire mtDNA of 28 Sardinian goats, selected to represent the widest possible diversity of the most widespread mitochondrial evolutionary lineage, haplogroup (Hg) A. These specimens were reporters of the diversity in the island but also elsewhere, as inferred from their affiliation to each of 11 clades defined by D-loop variation. Two reference sequences completed the dataset. Overall, 206 variations were found in the full set of 30 sequences, of which 23 were protein-coding non-synonymous single nucleotide substitutions. Many polymorphic sites within Hg A were informative for the reconstruction of its internal phylogeny. Bayesian and network clustering revealed a general similarity over the entire molecule of sequences previously assigned to the same D-loop clade, indicating evolutionarily meaningful lineages. Two major sister groupings emerged within Hg A, which parallel distinct geographical distributions of D-loop clades in extant stocks. The pattern of variation in protein-coding genes revealed an overwhelming role of purifying selection, with the quota of surviving variants approaching neutrality. However, a simple model of relaxation of selection for the bulk of variants here reported should be rejected. Non-synonymous diversity of Hg's A, B and C denoted that a proportion of variants not greater than that allowed in the wild was given the opportunity to spread into domesticated stocks. Our results also confirmed that a remarkable proportion of pre-existing Hg A diversity became incorporated into domestic stocks. Our results confirm clade A11 as a well differentiated and ancient lineage peculiar of Sardinia.

Identification of 2127 new HLA class I alleles in potential stem cell donors from Germany, the United States and Poland

We describe 2127 new human leukocyte antigen (HLA) class I alleles found in registered stem cell donors. These alleles represent 28.9% of the currently known class I alleles. Comparing new allele sequences to homologous sequences, we found 68.1% nonsynonymous nucleotide substitutions, 28.9% silent mutations and 3.0% nonsense mutations. Many substitutions occurred at positions that have not been known to be polymorphic before. A large number of HLA alleles and nucleotide variations underline the extreme diversity of the HLA system. Strikingly, 156 new alleles were found not only multiple times, but also in carriers of various parentage, suggesting that some new alleles are not necessarily rare. Moreover, new alleles were found especially often in minority donors. This emphasizes the benefits of specifically recruiting such groups of individuals.

Description of five novel HLA‐B alleles, B*07:184, B*41:27, B*42:19, B*50:32 and B*57:63, identified in Brazilian individuals

Five novel HLA-B alleles were identified by HLA-SBT typing in seven unrelated Brazilian individuals. The new alleles discovered include HLA-B*07:184, B*41:27, B*42:19, B*50:32 and B*57:63 and were officially named by the World Health Organization (WHO) Nomenclature Committee. All new HLA-B alleles had nonsynonymous nucleotide substitution polymorphisms when compared to their most closely related HLA-B allele.

Evolution of a Cell Culture-Derived Genotype 1a Hepatitis C Virus (H77S.2) during Persistent Infection with Chronic Hepatitis in a Chimpanzee

Persistent infection is a key feature of hepatitis C virus (HCV). However, chimpanzee infections with cell culture-derived viruses (JFH1 or related chimeric viruses that replicate efficiently in cell culture) have been limited to acute-transient infections with no pathogenicity. Here, we report persistent infection with chronic hepatitis in a chimpanzee challenged with cell culture-derived genotype 1a virus (H77S.2) containing 6 cell culture-adaptive mutations. Following acute-transient infection with a chimeric H77/JFH1 virus (HJ3-5), intravenous (i.v.) challenge with 10(6) FFU H77S.2 virus resulted in immediate seroconversion and, following an unusual 4- to 6-week delay, persistent viremia accompanied by alanine aminotransferase (ALT) elevation, intrahepatic innate immune responses, and diffuse hepatopathy. This first persistent infection with cell culture-produced HCV provided a unique opportunity to assess evolution of cell culture-adapted virus in vivo. Synonymous and nonsynonymous nucleotide substitution rates were greatest during the first 8 weeks of infection. Of 6 cell culture-adaptive mutations in H77S.2, Q1067R (NS3) had reverted to Q1067 and S2204I (NS5A) was replaced by T2204 within 8 weeks of infection. By 62 weeks, 4 of 6 mutations had reverted to the wild-type sequence, and all reverted to the wild-type sequence by 194 weeks. The data suggest H77S.2 virus has greater potential for persistence and pathogenicity than JFH1 and demonstrate both the capacity of a nonfit virus to persist for weeks in the liver in the absence of detectable viremia as well as strong selective pressure against cell culture-adaptive mutations in vivo. This study shows that mutations promoting the production of infectious genotype 1a HCV in cell culture have the opposite effect and attenuate replication in the liver of the only fully permissive animal species other than humans. It provides the only example to date of persistent infection in a chimpanzee challenged with cell culture-produced virus and provides novel insight into the forces shaping molecular evolution of that virus during 5 years of persistent infection. It demonstrates that a poorly fit virus can replicate for weeks within the liver in the absence of detectable viremia, an observation that expands current concepts of HCV pathogenesis and that is relevant to relapses observed with direct-acting antiviral therapies.

Allelic Frequency in Human SNPs Predicts the Rate of Non-Synonymous Nucleotide Substitutions between Human and Chimpanzee Genes

The combination of comparative genomics and population genetics may provide important clues regarding human evolution. We have hypothesized that the divergence between various human and chimpanzee orthologs will be reflected in the variability of single nucleotide polymorphisms (SNPs) that are localized in the vicinity of the corresponding loci in different human populations. Consistently with this notion, more diverged genes between humans and chimpanzees are more likely to be associated with human speciation and are anticipated to contain SNPs with reduced variability between different human populations. In order to test this hypothesis, we have compared the rate of non synonymous nucleotide substitutions (Ka) between 255 chimpanzee and human orthologs with the average deviation in the allelic frequencies of corresponding closely linked SNPs in two distinct human populations: The Yoruba people in Ibadan, Nigeria (YRI) and US residents with ancestry from Northern and Western Europe, collected in 1980 by the Centre d’Etude du Polymorphisme Humain (CEU). We found a significant (p

Positional cloning of the rice male sterility gene ms-IR36, widely used in the inter-crossing phase of recurrent selection schemes

The monogenetic recessive male-sterile gene ms-IR36 is widely used to facilitate the inter-crossing phase of recurrent selection in rice (Oryza sativa), but its segregation within the progeny disturbs other breeding phases. Marker-assisted early identification of msms and Msms seedlings would help overcome this drawback. Using successively bulked segregant analysis and large F2 populations, we mapped the ms-IR36 gene to a 33-kb region on the short arm of chromosome 2 that includes 10 candidate genes. Sequencing of these candidates together with checking rice genome annotations and expression databases allowed the target to be narrowed down to one candidate gene already isolated and characterized as the tapetum degeneration retardation (TDR) gene and reported to be involved in tapetal programmed cell death. Comparison of the sequence of the TDR gene between male-sterile (MS) and male-fertile (MF) IR36 plants detected one non-synonymous nucleotide substitution affecting the active domain of the encoded protein. Perfect co-segregation was observed between polymorphism at this nucleotide (SNP) and the MS/MF phenotype of 946 F2 plants. Spatial modelling of the active domain of the candidate protein reinforced the candidate status of the only SNP identified. Histological characterization of anther development in MS IR36 revealed defects identical to the ones observed in mutants used for the isolation and characterization of the TDR gene: delayed/non-degradation of tapetum tissue and collapse of the haploid microspores. We concluded that ms-IR36 corresponded to the TDR gene with a different mutation from the earlier one described in the same gene. No significant linkage drag was associated with ms-IR36. A SNP-based marker that enables simple early identification of MS plants and MF plants with the Msms genotype was designed.

Identification of the new HLA-DPB1 allele,DPB1*162:01, in a Venezuelan family

The allele HLA-DPB1*162:01 differs from DPB1*01:01:02 by three nonsynonymous nucleotide substitutions in exons 3 and 4.

Genetic analysis of G12P[8] rotaviruses detected in the largest U.S. G12 genotype outbreak on record

In 2006–07, 77 cases of gastroenteritis in Rochester, NY, USA were associated with rotavirus genotype G12P[8]. Sequence analysis identified a high degree of genetic relatedness among the VP7 and VP4 genes of the Rochester G12P[8] strains and between these strains and currently circulating human G12P[8] strains. Out of 77 samples, two and seven unique nucleotide sequences were identified for VP7 and VP4 genes, respectively. Rochester strain VP7 genes were found to occupy the G12-III lineage and VP4 genes clustered within the P[8]-3 lineage. Six strains contained non-synonymous nucleotide substitutions that produced amino acid changes at 6 sites in the VP8∗ region of the VP4 gene. Two sites (amino acids 242 and 246) were located in or near a described trypsin cleavage site. Selection analyses identified one positively selected VP7 site (107) and strong purifying selection at 58 sites within the VP7 gene as well as 2 of the 6 variant sites (79 and 218) in VP4.

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

BackgroundThe twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence.Methodology/Principal FindingsThe aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species.Conclusions/SignificanceTatC proteins appear to be diversifying within particular bacterial classes and its specialization may be driven by the substrates it transports and the environment of its host.

The variability of the large genomic segment of Ťahyňa orthobunyavirus and an all-atom exploration of its anti-viral drug resistance

Ťahyňa virus (TAHV), a member of the Bunyaviridae family (California complex), is an important but neglected human mosquito-borne pathogen. The virus genome is composed of three segments, i.e., small (S), medium (M), and large (L). Previous studies on genetic variability of viruses within the California complex were focused on S and M segments, but the L segment remains relatively unstudied. To assess the genetic variation and the relation to virus phenotype we analyzed the L segment sequences of biologically diverse TAHV strains isolated in the Czech Republic and Slovakia. Phylogenetic analysis covering all available sequences of the L segment of TAHV clearly revealed two distinguished lineages, tentatively named as “European” and “Asian”. The L segment strains within the European lineage are highly conserved (identity 99.3%), whilst Asian strains are more genetically diverse (identity 97%). Based on sequence comparison with other bunyaviruses, several non-synonymous nucleotide substitutions unique for TAHV in the L segment were identified. We also identified specific residue substitutions in the endonuclease domain of TAHV compared with the La Crosse virus. Since the endonuclease domain of the La Crosse virus has been resolved, we employed an all energy landscape algorithm to analyze the ligand migration of a viral polymerase inhibitor. This allowed us to demonstrate, at the atomic level, that this viral polymerase inhibitor randomly explored the specific residue substitutions in the endonuclease domain of the TAHV L segment.

Phenotypic and genotypic characterization of induced acyclovir-resistant clinical isolates of herpes simplex virus type 1

Eleven strains of acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) were generated from HSV-1 clinical isolates by exposure to ACV. Genotype of the thymidine kinase (TK) and DNA polymerase (pol) genes from these mutants were further analyzed. Genotypic analysis revealed four non-synonymous mutations in TK gene associated with gene polymorphism and two to three non-synonymous mutations in DNA pol gene. Seven and six strains contained at least one resistance-associated mutation at TK and DNA pol gene, respectively. Resistance-associated mutations within the TK gene consisted of 64% of non-synonymous frameshift mutations within the homopolymer region of G’s and C’s, and 36% of non-synonymous nucleotide substitutions of the conserved gene region (C336Y, R51W and R222H), nucleotide that produced stop codon (L288Stop) and two amino acid substitutions outside the conserved region (E39G & L208F). There were 10 non-synonymous amino acid substitutions located outside the conserved region with the unclear significance to confer resistance observed. Resistance-associated mutations in DNA pol gene include insertion of G at the homopolymer region of G’s (794–797) and amino acid substitutions inside (V621S) or outside (H1228D) the conserved region. In silico analysis of the mutated TK (C336Y, R51W and L208F), and DNA pol (V621S and H1228D) suggested structural changes that might alter the stability of these proteins. However, there were several mutations with unclear significance to confer ACV-resistance identified, especially mutations outside the conserved region.

Polymorphisms in major histocompatibility complex class IIα genes are associated with resistance to infectious hematopoietic necrosis in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792)

Summary Infectious hematopoietic necrosis is a serious viral disease of salmonids, including rainbow trout Oncorhynchus mykiss, and causes tremendous economic losses to the rainbow trout farming industry. Major histocompatibility complex (MHC) genes are crucial elements of adaptive immunity in vertebrate organisms and have been linked with the resistance to numerous pathogenic diseases. In this study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by cloning and sequencing were used to examine polymorphisms in the DAA genes (specifically DAA exon 2 of MHC class IIα) of rainbow trout and investigate their association with the infectious hematopoietic necrosis virus (IHNV) resistance in rainbow trout. Seventeen alleles were resolved, including 13 novel alleles. Individuals possessed between two and five alleles, indicating that the genome harbours at least three closely-related DAA exon 2 loci. The ratio of non-synonymous to synonymous nucleotide substitutions suggested that DAA exon 2 is under positive selection. A greater variability of amino acids and non-synonymous nucleotide substitution rate was evident in the peptide-binding region (PBR) than in the non-PBR (27.75%). Importantly, the analyses revealed that certain MHC class IIα alleles appear to confer resistance to IHNV in rainbow trout, while others confer susceptibility. The most common alleles in the resistant populations of rainbow trout, Onmy-DAA*1301 and Onmy-DAA*0304, confer resistance to IHNV and were not present in the susceptible population. Hence, these alleles may be ideal molecular markers that can assist the breeding of IHNV resistance in rainbow trout.

Phylogenetic relationships among Perissodactyla: Secretoglobin 1A1 gene duplication and triplication in the Equidae family

Secretoglobin family 1A member 1 (SCGB 1A1) is a small anti-inflammatory and immunomodulatory protein that is abundantly secreted in airway surface fluids. We recently reported the existence of three distinct SCGB1A1 genes in the domestic horse genome as opposed to the single gene copy consensus present in other mammals. The origin of SCGB1A1 gene triplication and the evolutionary relationship of the three genes amongst Equidae family members are unknown. For this study, SCGB1A1 genomic data were collected from various Equus individuals including E. caballus, E. przewalskii, E. asinus, E. grevyi, and E. quagga. Three SCGB1A1 genes in E. przewalskii, two SCGB1A1 genes in E. asinus, and a single SCGB1A1 gene in E. grevyi and E. quagga were identified. Sequence analysis revealed that the non-synonymous nucleotide substitutions between the different equid genes coded for 17 amino acid changes. Most of these changes localized to the SCGB 1A1 central cavity that binds hydrophobic ligands, suggesting that this area of SCGB 1A1 evolved to accommodate diverse molecular interactions. Three-dimensional modeling of the proteins revealed that the size of the SCGB 1A1 central cavity is larger than that of SCGB 1A1A. Altogether, these findings suggest that evolution of the SCGB1A1 gene may parallel the separation of caballine and non-caballine species amongst Equidae, and may indicate an expansion of function for SCGB1A1 gene products.

A new allele, HLA‐DQB1*04:09

The novel allele HLA-DQB1*04:09 differs from DQB1*04:02:01 by three nonsynonymous nucleotide substitutions in exon 2.

Molecular characterization and expression profile of ghrelin gene during different reproductive phases in buffalo (Bubalus bubalis)

Ghrelin, a novel motilin-related endogenous ligand for growth hormone secretagouge receptor, is implicated in various biological functions, including regulation of female reproduction. But the presence of ghrelin and its role in reproductive functions in buffalo, a species with poor reproductive efficiency, is not known. In the present study full-length ghrelin cDNA was isolated from bubaline abomasum, which encodes the entire prepropeptide of 116 amino acids. The deduced amino acid sequence of ghrelin of buffalo showed >95% and 31% identity with that of ruminants (cattle, sheep, and goat) and humans, respectively. Analysis of synonymous and nonsynonymous nucleotide substitutions in the coding region of ghrelin indicated that these sequences of different species have been under purifying selection. The 3995-bp amplicon of ghrelin gene consisting of 4 exons and 3 introns was cloned with genomic DNA from buffalo. Further, ghrelin expression was determined by quantitative real-time PCR, in situ hybridization, and immunohistochemistry in bubaline endometrial tissues at different stages of the estrous cycle and early pregnancy. Our results indicated the persistent expression of ghrelin mRNA and protein in the endometrium during stage I (day 3–5), stage II (day 6–15), and stage III (day 16–21) of the estrous cycle and also during early (∼day 30–40) pregnancy. Immunohistochemistry and quantitative real-time PCR experiments indicated the relatively higher expression of ghrelin in the endometrium during stage II (day 6–15) of the estrous cycle and early pregnancy than during stage I (day 3–5) and stage III (day 16–21) of the estrous cycle, but no statistically significant difference in ghrelin expression was observed among stages. To conclude, the results of the present study indicate the persistent expression of ghrelin in the uterine endometrium throughout the estrous cycle and in early pregnancy which might be helpful in determining its role in buffalo reproduction.