Coding Sequence Of Gene Research Articles

The chromosome-level Elaeagnus mollis genome and transcriptomes provide insights into genome evolution, glycerolipid and vitamin E biosynthesis in seeds

Elaeagnus mollis, which has seeds with high lipid and vitamin E contents, is a valuable woody oil plant with potential for utilization. Currently, the biosynthesis and regulation mechanism of glycerolipids and vitamin E are still unknown in E. mollis. Here, we present the chromosome-level reference genome of E. mollis (scaffold N50: ~40.66Mbp, genome size: ~591.48Mbp) by integrating short-read, long-read, and Hi-C sequencing platforms. A total of 36,796 protein-coding sequences, mainly located on 14 proto-chromosomes, were predicted. Additionally, two whole genome duplication (WGD) events were suggested to have occurred ~54.07 and ~35.06 million years ago (MYA), with Elaeagnaceae plants probably experiencing both WGD events. Furthermore, the long terminal retrotransposons in E. mollis were active ~0.23MYA, and one of them was inferred to insert into coding sequence of the negative regulatory lipid synthesis gene, EMF2. Through transcriptomic and metabonomic analysis, key genes contributing to the high lipid and vitamin E levels of E. mollis seeds were identified, while miRNA regulation was also considered. This comprehensive work on the E. mollis genome not only provides a solid theoretical foundation and experimental basis for the efficient utilization of seed lipids and vitamin E, but also contributes to the exploration of new genetic resources.

Primary Sequence and Three-Dimensional Structural Comparison between Malanin and Ricin, a Type II Ribosome-Inactivating Protein.

Malanin is a new type II ribosome-inactivating protein (RIP) purified from Malania oleifera, a rare, endangered tree is only found in the southwest of Guangxi Province and the southeast of Yunnan Province, China. The gene coding sequence of malanin was found from the cDNA library of M. oleifera seeds by employing the ten N-terminal amino acid sequences of malanin, DYPKLTFTTS for chain-A and DETXTDEEFN (X was commonly C) for chain-B. The results showed a 65% amino acid sequence homology between malanin and ricin by DNAMAN 9.0 software, the active sites of the two proteins were consistent, and the four disulfide bonds were in the same positions. The primary sequence and three-dimensional structures of malanin and ricin are likely to be very similar. Our studies suggest that the mechanism of action of malanin is expected to be analogous to ricin, indicating that it is a member of the type II ribosome-inactivating proteins. This result lays the foundation for further study of the anti-tumor activities of malanin, and for the application of malanin as a therapeutic agent against cancers.

8415 Rapid Generation Of An In Vivo Loss-Of-Function Genetic Model Of sdhb Zebrafish: Functional And Metabolic Characterization

Abstract Disclosure: S. Parisien-La Salle: None. F. Nobilleau: None. J. Lamontagne: None. S. Éric: None. I. Bourdeau: None. Introduction: Animal research has been limited in pheochromocytomas and paragangliomas (PPGLs). Recently, Dona et al., described a sdhb mutated zebrafish model by introducing a 13 bp frameshift mutation at the sdhb exon1—intron1 boundary into the zebrafish sdhb gene that recapitulated features of PPGLs. Objective: To generate a new model of mutated sdhb zebrafish to further investigate the underlying metabolic and biochemical perturbations in vivo. Methods: We took advantage of the genetic accessibility of zebrafish embryo to generate loss-of-function model for sdhb using CRISPR/Cas9 technology. Three guide RNAs were designed and microinjected in one-cell stage zebrafish embryos to target the coding sequence of zebrafish sdhb gene. In order to control for the stress brought on by injections, we injected a group of zebrafish with Cas9 endonuclease as a control. At five days post fertilization, mutant and control zebrafish were flash frozen and sent for liquid chromatography mass spectrometry measurements of Krebs cycle metabolites and normetanephrines/metanephrines. All results were normalized to the control groups consisting of wild-type or Cas9 injected larvae. Sdhb expression was analyzed by RT-qPCR in sdhb mutants and wild-type zebrafish larvae. We also monitored survival and heart rate in sdhb mutant zebrafish, compared to wild-type and Cas9. Results: Firstly, we confirmed the significant reduction of sdhb expression in CRISPR-injected larvae compared to wild-type larvae at 5 dpf (0.0613 vs 1.000 p<0.0001). Our metabolic panel showed that succinate was up to 26 times more elevated in sdhb mutants than in wild-type fish extracts (sdhb: 26.51 p<0.0001). Lactate (sdhb: 5.27 p<0.0001), leucine (sdhb: 2.88 p<0.0001), arginine (sdhb: 1.50 p=0.0034) and HMG-CoA (sdhb: 1.36 p=0.0033) were also higher in sdhb mutants. Whereas aspartate (sdhb: 0.244 p<0.0001) and oxidized gluthiatone (sdhb: 0.64 p=0.0011) were significantly lower in sdhb mutants than in wild-types. In regards to functionality, normetanephrine and metanephrine levels were at least four folds higher in sdhb mutants than in Cas9 injected larvaes (4.06 p<0.0001 and 3.42 p<0.0001 respectively). Interestingly, this difference was more noticeable in the fish bathing medium than in larvae tissue extracts, suggesting an increased hormonal excretion (normetanephrines: 4.81 p<0.0001 and metanephrines: 9.40 p<0.0001). Finally, sdhb mutated zebrafish presented with a higher heart rate (148 vs 133 p<0.0001) and reduced lifespan (p<0.0001) when compared to the wild-type/Cas9 group. Conclusion: These findings confirm that the zebrafish is an ideal model for studying PPGLs and their associated genetic defects. Our approach allows the in vivo assessment of a large metabolic panel, opening avenues on new potential clinical metabolic biomarkers in a loss-of-function of the SDHB gene. Ref: Dona M, et al. Endocr Relat Cancer. 2021 Presentation: 6/1/2024

Genetic variation of FUT3 gene in the Han population from Northern China.

The FUT3 gene encodes α(1,3/1,4)-fucosyltransferase, which is a crucial enzyme in the synthesis of Lewis antigens. FUT3 gene variants show race-specific differences. In this study, we conducted a systematic sequence analysis of the FUT3 coding sequence. The objective was to explore genetic variations of the FUT3 gene within the Han population of Northern China. A cohort of 313 blood donors was recruited for the study. The coding sequence of the FUT3 gene was amplified using polymerase chain reaction, followed by sequencing and haplotype construction. Twelve single nucleotide variations (SNVs) were identified within the coding sequence of the FUT3 gene. Notably, the c.59 T > G site exhibited the highest mutation frequency of 43.13%, followed by the c.508G > A and c.1067 T > A sites with mutation frequencies of 27.48% and 16.93%, respectively. Le was the most common haplotype, accounting for 67.57% of the cases, and Le/Le was the most common diplotype, accounting for 46.33% of the cases. The study also highlighted a significant difference in mutation frequencies of FUT3 gene between the Han Chinese of Northern China and the Dai of Xishuangbanna, China, but not the Han Chinese in Beijing in the North and the Southern Han Chinese, emphasising that Han Chinese in Northern China are genetically most distant from Europeans and closest to East Asians. Our study characterises FUT3 gene variations in Han Chinese from Northern China, and provides basic genetic data for genetics, forensic medicine, and genotyping of Lewis blood groups.

Mechanical and structural features of three AcSp proteins underlie the diverse material properties of aciniform silks of Neoscona spiders

Spider silks are desirable multicomponent biomaterials characterized by great tensile strength, extensibility, and biocompatibility. Of all spider silk types, aciniform silk has highest toughness due to its combination of high tensile strength and elsticity. Here, we identify three major spidroin components (AcSp1A, AcSp1B, and AcSp2) from aciniform silk of orbweb weaving spider, Neoscona scylloides, and present their full-length coding gene sequences. Comparative sequence and expression level analysis show that AcSp1B has highest expression level and higher serine content than other two AcSp proteins, while the AcSp2 shows very low mRNA level. Furthermore, three recombinant minimalist AcSp proteins are produced and could be induced to form fibers by shear forces in a physiological buffer. The manual-drawn AcSp1B fiber shows strongest tensile strength among three AcSp fibers because of its higher β-sheet formed by abundant serine content. We also compare mechanical properties of aciniform silks between two Neoscona species (N. theisi and N. scylloides) and found that aciniform silks from N. theisi exhibit higher tensile strength than those of N. scylloides, which may result from altering expression levels of two AcSp1 proteins. Collectively, our results provide insights into the mechanical features of each component in aciniform silk from N. scylloides and reveal the molecular mechanism of diverse material properties of aciniform silk among species.

Customizable gene sensing and response without altering endogenous coding sequences.

Synthetic biology aims to modify cellular behaviors by implementing genetic circuits that respond to changes in cell state. Integrating genetic biosensors into endogenous gene coding sequences using clustered regularly interspaced short palindromic repeats and Cas9 enables interrogation of gene expression dynamics in the appropriate chromosomal context. However, embedding a biosensor into a gene coding sequence may unpredictably alter endogenous gene regulation. To address this challenge, we developed an approach to integrate genetic biosensors into endogenous genes without modifying their coding sequence by inserting into their terminator region single-guide RNAs that activate downstream circuits. Sensor dosage responses can be fine-tuned and predicted through a mathematical model. We engineered a cell stress sensor and actuator in CHO-K1 cells that conditionally activates antiapoptotic protein BCL-2 through a downstream circuit, thereby increasing cell survival under stress conditions. Our gene sensor and actuator platform has potential use for a wide range of applications that include biomanufacturing, cell fate control and cell-based therapeutics.

Complete genome sequence of Streptococcus intermedius strain XH2169.

Here, we report the complete genome sequence of Streptococcus intermedius strain XH2169 isolated from a blood sample in China. The genome comprises a single circular chromosome with a length of 1,944,282 bp. It harbored 1,891 coding gene sequences, 16 rRNA genes, 60 tRNA genes, and 3 noncoding RNA genes.

Genome-wide characterization of extrachromosomal circular DNA in SLE and functional analysis reveal their association with apoptosis

Extrachromosomal circular DNA (eccDNA) derived from linear chromosomes, are showed typical nucleosomal ladder pattern in agarose gel which as a known feature of apoptosis and demonstrated to be immunogenicity. In systemic lupus erythematosus (SLE) patients, elevated levels of cell-free DNA (cfDNA) can be found in either linear forms or circular forms, while circular ones are much less common and harder to detect. The molecular characteristics and function of circular forms in plasma SLE patients remains elusive. Herein, we characterized the hallmarks of plasma eccDNA in SLE patients, including the lower normalized number and GC content of eccDNA in SLE plasma than in the healthy, and SLE eccDNA number positively correlated with C3 and negatively with anti-dsDNA antibodies. The differential eccGenes (eccDNAs carrying the protein coding gene sequence) of SLE was significantly enriched in apoptosis-related pathways. The artificially synthesized eccDNA with sequences of the PRF1 exon region could promote transcriptional expression of PRF1, IFNA and IFIT3 and inhibit early-stage apoptosis. Plasma eccDNA can serve as a novel autoantigen in the pathogenesis of SLE.

MS2/GmAMS1 encodes a bHLH transcription factor important for tapetum degeneration in soybean.

GmAMS1 is the only functional AMS and works with GmTDF1-1 and GmMS3 to orchestrate the tapetum degeneration in soybean. Heterosis could significantly increase the production of major crops as well as soybean [Glycine max (L.) Merr.]. Stable male-sterile/female-fertile mutants including ms2 are useful resources to apply in soybean hybrid production. Here, we identified the detailed mutated sites of two classic mutants ms2 (Eldorado) and ms2 (Ames) in MS2/GmAMS1 via the high-throughput sequencing method. Subsequently, we verified that GmAMS1, a bHLH transcription factor, is the only functional AMS member in soybean through the complementary experiment in Arabidopsis; and elucidated the dysfunction of its homolog GmAMS2 is caused by the premature stop codon in the gene's coding sequence. Further qRT-PCR analysis and protein-protein interaction assays indicated GmAMS1 is required for expressing downstream members in the putative DYT1-TDF1-AMS-MYB80/MYB103/MS188-MS1 cascade module, and might regulate the upstream members in a feedback mechanism. GmAMS1 could interact with GmTDF1-1 and GmMS3 via different region, which contributes to dissect the mechanism in the tapetum degeneration process. Additionally, as a core member in the conserved cascade module controlling the tapetum development and degeneration, AMS is conservatively present in all land plant lineages, implying that AMS-mediated signaling pathway has been established before land plants diverged, which provides further insight into the tapetal evolution.

Identification of metal tolerance proteins (MTP) and their gene expression under drought stress in potato (Solanum tuberosum L.)

Metal tolerance proteins (MTPs) are as metal efflux transporters, existing extensively at all plant sections and play significant roles in regulation of the metal levels in biological processes. In the current study, phylogenetic relationships, gene structures, conserved motifs, and StMTP domains were analyzed. Here, 12 MTP genes in S. tuberosum were detected and categorized in three major clusters namely Fe/Zn-MTP, Zn-MTP, and Mn-MTP and seven groups (1, 5, 6, 7, 8, 9, and 11) according to phylogenetic relationships. Based on in silico and qPCR analysis, all of StMTPs included a cation diffusion facilitator (CDF) domains and the putative Mn-MTP harbored the ZT-dimer. An evolutionary analysis indicated that StMTP genes had undergone gene duplication leading to gene loss and gene expansion events. Analysis of transcription factor binding sites (TFBS) and microRNA in promoter region and coding sequence of StMTP genes revealed the presence of 5312 putative TFBS and 13 StmiRNAs. The analysis of promoter regions of StMTP genes possess various frequencies of TFBS, illustrating various responses in different growth and developmental stages as well as under abiotic stress. Expression profile analysis revealed that the StMTP9 were up-regulated in leaves and stem, while, StMTP8 up-regulated in leaves. Both genes down-regulated in tubers, roots as well as under drought stress. These results will provide a better insight for functional characterization of StMTP genes and can be helpful to elucidate the biological structure of their genes in potato.

Unveiling the mysteries of HvANS: a study on anthocyanin biosynthesis in qingke (hordeum vulgare L. var. Nudum hook. f.) seeds

BackgroundBased on our previous research, a full-length cDNA sequence of HvANS gene was isolated from purple and white Qingke. The open reading frame (ORF) in the purple variety Nierumuzha was 1320 base pairs (bp), encoding 439 amino acids, while the ORF in the white variety Kunlun 10 was 1197 bp, encoding 398 amino acids. A nonsynonymous mutation was found at the position of 1195 bp (T/C) in the coding sequence (CDS) of the HvANS gene. We carried out a series of studies to further clarify the relationship between the HvANS gene and anthocyanin synthesis in Qingke.ResultsThe conservative structural domain prediction results showed that the encoded protein belonged to the PLN03178 superfamily. Multiple comparisons showed that this protein had the highest homology with Hordeum vulgare, at 88.61%. The approximately 2000 bp promoter sequence of the HvANS gene was identical in both varieties. The real-time fluorescence PCR (qRT-PCR) results revealed that HvANS expression was either absent or very low in the roots, stems, leaves, and awns of Nierumuzha. In contrast, the HvANS expression was high in the seed coats and seeds of Nierumuzha. Likewise, in Kunlun 10, HvANS expression was either absent or very low, indicating a tissue-specific and variety-specific pattern for HvANS expression. The subcellular localization results indicated that HvANS was in the cell membrane. Metabolomic results indicated that the HvANS gene is closely related to the synthesis of three anthocyanin substances (Idaein chloride, Kinetin 9-riboside, and Cyanidin O-syringic acid). Yeast single hybridization experiments showed that the HvANS promoter interacted with HvANT1, which is the key anthocyanin regulatory protein. In a yeast two-hybrid experiment, we obtained two significantly different proteins (ZWY2020 and POMGNT2-like) and verified the results by qRT-PCR.ConclusionsThese results provide a basis for further studies on the regulatory mechanism of HvANS in the synthesis of anthocyanins in Qingke purple grains.

Expression Analysis and Interaction Protein Screening of CoGI, the Key Factor in Photoperiod Regulation of Flowering in Camellia oleifera Abel

Photoperiod is a pivotal regulatory factor in the flowering of Camellia oleifera Abel. (C. oleifera). GIGANTEA (GI) serves as a pivotal regulator, not only orchestrating the intricate circadian rhythm but also governing photoperiod-dependent flowering. In order to explore the function of GI in C. oleifera (CoGI), we obtained a CoGI gene-coding sequence and analyzed a CoGI protein sequence using bioinformatics. Furthermore, we conducted a spatiotemporal expression analysis of CoGI. And a yeast two-hybridization assay was used to screen the interacting proteins of CoGI. Evolutionary analysis revealed high conservation of the CoGI protein, which clustered with the GI protein from Camellia sinensis (CsGI) on a common evolutionary branch. The expression of CoGI was different in each part, and a tissue expression analysis revealed that the relative expression level of the CoGI gene is highest in the leaves of C. oleifera, while it is at its lowest in the seed coats. Transgenic Arabidopsis thaliana (Arabidopsis) overexpressing CoGI exhibited early flowering under long-day conditions. In addition, the yeast two-hybrid library screening revealed interactions between seven C. oleifera proteins and CoGI: CoACR9, CoLAO, CoDExH12-like, CoIT1K-like, CoUPF0481, CoIDM3, and CoAt4g27190-like. The findings demonstrated that CoGI is crucial to C. oleifera’s flowering.

The transcriptional regulation of the horizontally acquired iron uptake system, yersiniabactin and its contribution to oxidative stress tolerance and pathogenicity of globally emerging salmonella strains

ABSTRACT The bacterial species Salmonella enterica (S. enterica) is a highly diverse pathogen containing more than 2600 distinct serovars, which can infect a wide range of animal and human hosts. Recent global emergence of multidrug resistant strains, from serovars Infantis and Muenchen is associated with acquisition of the epidemic megaplasmid, pESI that augments antimicrobial resistance and pathogenicity. One of the main pESI’s virulence factors is the potent iron uptake system, yersiniabactin encoded by fyuA, irp2-irp1-ybtUTE, ybtA, and ybtPQXS gene cluster. Here we show that yersiniabactin, has an underappreciated distribution among different S. enterica serovars and subspecies, integrated in their chromosome or carried by different conjugative plasmids, including pESI. While the genetic organization and the coding sequence of the yersiniabactin genes are generally conserved, a 201-bp insertion sequence upstream to ybtA, was identified in pESI. Despite this insertion, pESI-encoded yersiniabactin is regulated by YbtA and the ancestral Ferric Uptake Regulator (Fur), which binds directly to the ybtA and irp2 promoters. Furthermore, we show that yersiniabactin genes are specifically induced during the mid-late logarithmic growth phase and in response to iron-starvation or hydrogen peroxide. Concurring, yersiniabactin was found to play a previously unknown role in oxidative stress tolerance and to enhance intestinal colonization of S. Infantis in mice. These results indicate that yersiniabactin contributes to Salmonella fitness and pathogenicity in vivo and is likely to play a role in the rapid dissemination of pESI among globally emerging Salmonella lineages.

Genomic analyses of withers height and linear conformation traits in German Warmblood horses using imputed sequence-level genotypes

BackgroundBody conformation, including withers height, is a major selection criterion in horse breeding and is associated with other important traits, such as health and performance. However, little is known about the genomic background of equine conformation. Therefore, the aim of this study was to use imputed sequence-level genotypes from up to 4891 German Warmblood horses to identify genomic regions associated with withers height and linear conformation traits. Furthermore, the traits were genetically characterised and putative causal variants for withers height were detected.ResultsA genome-wide association study (GWAS) for withers height confirmed the presence of a previously known quantitative trait locus (QTL) on Equus caballus (ECA) chromosome 3 close to the LCORL/NCAPG locus, which explained 16% of the phenotypic variance for withers height. An additional significant association signal was detected on ECA1. Further investigations of the region on ECA3 identified a few promising candidate causal variants for withers height, including a nonsense mutation in the coding sequence of the LCORL gene. The estimated heritability for withers height was 0.53 and ranged from 0 to 0.34 for the conformation traits. GWAS identified significantly associated variants for more than half of the investigated conformation traits, among which 13 showed a peak on ECA3 in the same region as withers height. Genetic parameter estimation revealed high genetic correlations between these traits and withers height for the QTL on ECA3.ConclusionsThe use of imputed sequence-level genotypes from a large study cohort led to the discovery of novel QTL associated with conformation traits in German Warmblood horses. The results indicate the high relevance of the QTL on ECA3 for various conformation traits, including withers height, and contribute to deciphering causal mutations for body size in horses.

Molecular characterization, function, tissue differential expression, and single-nucleotide polymorphism of buffalo TP53 gene

Abstract. TP53 has been shown to be involved in lactation in cattle. However, the role of TP53 in buffalo lactation remains unknown. To this end, we isolated and identified the complete coding sequence (CDS) of the TP53 gene from the buffalo mammary gland and further analyzed its molecular characteristics, function, tissue differential expression, and single-nucleotide polymorphism (SNP). A transcript of this gene was cloned with a CDS length of 1161 bp, encoding a protein consisting of 386 amino acid residues. Bioinformatics analysis showed that buffalo TP53 CDS and the physicochemical characteristics, conserved domains, structure, and function of its encoded protein are highly similar to those of other species in Bovidae. The buffalo TP53 protein contains an N-terminal activation domain, a DNA-binding domain, and a tetrameric domain, and it plays a functional role in the nucleus. TP53 was found to express in all 11 detected buffalo tissues, and its expression in the heart, kidney, brain, muscle, and rumen during lactation was significantly higher than that during non-lactation (p<0.05), while in the liver, lung, and mammary gland, its expression was the opposite (p<0.05). Interference experiments in buffalo mammary epithelial cells (BuMECs) showed that TP53 inhibits the expression of genes related to milk protein and milk fat synthesis through the PI3K–AKT–mTOR pathway. A synonymous nucleotide substitution (c.204C > T) was found in the TP53 CDS of river buffalo, which is the CC homozygote in swamp buffalo. The results indicate that the TP53 gene is involved in buffalo lactation by negatively regulating the synthesis of milk protein and milk fat.

Clustering of predicted loss-of-function variants in genes linked with monogenic disease can explain incomplete penetrance

BackgroundGenetic variants that severely alter protein products (e.g. nonsense, frameshift) are often associated with disease. For some genes, these predicted loss-of-function variants (pLoFs) are observed throughout the gene, whilst in others, they occur only at specific locations. We hypothesised that, for genes linked with monogenic diseases that display incomplete penetrance, pLoF variants present in apparently unaffected individuals may be limited to regions where pLoFs are tolerated. To test this, we investigated whether pLoF location could explain instances of incomplete penetrance of variants expected to be pathogenic for Mendelian conditions.MethodsWe used exome sequence data in 454,773 individuals in the UK Biobank (UKB) to investigate the locations of pLoFs in a population cohort. We counted numbers of unique pLoF, missense, and synonymous variants in UKB in each quintile of the coding sequence (CDS) of all protein-coding genes and clustered the variants using Gaussian mixture models. We limited the analyses to genes with ≥ 5 variants of each type (16,473 genes). We compared the locations of pLoFs in UKB with all theoretically possible pLoFs in a transcript, and pathogenic pLoFs from ClinVar, and performed simulations to estimate the false-positive rate of non-uniformly distributed variants.ResultsFor most genes, all variant classes fell into clusters representing broadly uniform variant distributions, but genes in which haploinsufficiency causes developmental disorders were less likely to have uniform pLoF distribution than other genes (P < 2.2 × 10−6). We identified a number of genes, including ARID1B and GATA6, where pLoF variants in the first quarter of the CDS were rescued by the presence of an alternative translation start site and should not be reported as pathogenic. For other genes, such as ODC1, pLoFs were located approximately uniformly across the gene, but pathogenic pLoFs were clustered only at the end, consistent with a gain-of-function disease mechanism.ConclusionsOur results suggest the potential benefits of localised constraint metrics and that the location of pLoF variants should be considered when interpreting variants.

Identification of SNPs in the MAP4K4 Gene Coding Sequence and Their Association with Mastitis Susceptibility/Resistance in Nili Ravi Buffaloes

Identification of SNPs in the MAP4K4 Gene Coding Sequence and Their Association with Mastitis Susceptibility/Resistance in Nili Ravi Buffaloes

A population genomics analysis of the Aotearoa New Zealand endemic rewarewa tree (Knightia excelsa)

Rewarewa (Knightia excelsa, Proteaceae) is a tree species endemic to Aotearoa New Zealand, with a natural distribution spanning Te Ika-a-Māui (North Island) and the top of Te Waipounamu (South Island). We used the pseudo-chromosome genome assembly of rewarewa as a reference and whole genome pooled sequencing from 35 populations sampled across Aotearoa New Zealand, including trees growing on Māori-owned land, to identify 1,443,255 single nucleotide polymorphisms (SNPs). Four genetic clusters located in the northern North Island (NNI), eastern North Island (NIE), western and southern North Island (NIWS), and the South Island (SI) were identified. Gene flow was revealed between the SI and NIE genetic clusters, plus bottleneck and contraction events within the genetic clusters since the mid-late Pleistocene, with divergence between North and South Island clusters estimated to have occurred ~115,000–230,000 years ago. Genotype environment analysis (GEA) was used to identify loci and genes linked with altitude, soil pH, soil carbon, slope, soil size, annual mean temperature, mean diurnal range, isothermality, annual precipitation, and precipitation seasonality. The location of the SNPs associated with these environmental variables was compared with the position of 52,192 gene-coding sequences that were predicted in the rewarewa genome using RNA sequencing. This new understanding of the genetic variation present in rewarewa and insights into the genetic control of adaptive traits will inform efforts to incorporate the species in restoration plantings and for marketing rewarewa honey based on provenance.

Creation of Knock-In Alleles of Insulin Receptor Tagged by Fluorescent Proteins mCherry or EYFP in Fruit Fly Drosophila melanogaster.

The insulin/insulin-like growth factor-like signaling (IIS) pathway is highly conserved across metazoans and regulates numerous physiological functions, including development, metabolism, fecundity, and lifespan. The insulin receptor (InR), a crucial membrane receptor in the IIS pathway, is known to be ubiquitously expressed in various tissues, albeit at generally low levels, and its subcellular localization remains incompletely characterized. In this study, we employed CRISPR-mediated mutagenesis in the fruit fly Drosophila to create knock-in alleles of InR tagged with fluorescent proteins (InR::mCherry or InR::EYFP). By inserting the coding sequence of the fluorescent proteins mCherry or EYFP near the end of the coding sequence of the endogenous InR gene, we could trace the natural InR protein through their fluorescence. As an example, we investigated epithelial cells of the male accessory gland (AG), an internal reproductive organ, and identified two distinct patterns of InR::mCherry localization. In young AG, InR::mCherry accumulated on the basal plasma membrane between cells, whereas in mature AG, it exhibited intracellular localization as multiple puncta, indicating endocytic recycling of InR during cell growth. In the AG senescence accelerated by the mutation of Diuretic hormone 31 (Dh31), the presence of InR::mCherry puncta was more pronounced compared to the wild type. These findings raise expectations for the utility of the newly created InR::mCherry/EYFP alleles for studying the precise expression levels and subcellular localization of InR. Furthermore, this fluorescently tagged allele approach can be extended to investigate other membrane receptors with low abundance, facilitating the direct examination of their true expression and localization.

Comparison of Five Escherichia coli Strains to Achieve the Maximum Yield of a Recombinant Immunotoxin Consisting of an Antibody against VEGF Conjugated with MAP30 Toxin in a Benchtop Bioreactor.

Cancer is among the leading causes of death worldwide, imposing high costs on the health systems of all societies. Extensive biological studies are required to discover appropriate therapies. Escherichia coli has long been regarded as one of the main biotechnological bio-factories to produce recombinant protein-based therapeutics. In the present study, five strains of E. coli were compared to achieve the maximum production of a previously designed recombinant immunotoxin-carrying MAP30 toxin against VEGF-overexpressed cancer cells in a benchtop bioreactor. The recombinant immunotoxin coding gene sequence was extracted from the NCBI database. The host used to produce the recombinant immunotoxin were five E. coli strains of BL21 (DE3), DH5α, SHuffle®T7, XL1-Blue, and Rosetta-gamiTM (DE3). CaCl2 method was used for bacterial transformation. Bacterial growth measurements were performed using optical density measurements at 600 nm. The immunotoxin production was measured using SDS-PAGE analysis. The best-producing strain was cultivated in a 10-L benchtop stirred tank bioreactor. Recent patents on this field were also studied. The results demonstrated that the BL21 (DE3) strain had the highest expression of recombinant protein in comparison to other strains. Moreover, the cell growth of E. coli BL21 (DE3) and SHuffle®T7 strains before transformation in the LB medium, were significantly higher in comparison to other strains. Additionally, the transformation of Rosettagami was associated with decreased cell proliferation. The transformation of the XL1-Blue strain did not effect cell growth. Analysis of the growth kinetics demonstrated appropriate proliferation of the transformed BL21 (DE3) cells in the laboratory benchtop bioreactor. Based on the results of this study, the BL21 (DE3) strain could be used as a suitable host for the production of the recombinant immunotoxin against VEGF in stirred tank bioreactor, which can be employed for the treatment of tumors. Yet, its precise mechanism must be explored in extensive studies.