Structural Maintenance Of Chromosomes Research Articles

New insights into genome folding by loop extrusion from inducible degron technologies.

Chromatin folds into dynamic loops that often span hundreds of kilobases and physically wire distant loci together for gene regulation. These loops are continuously created, extended and positioned by structural maintenance of chromosomes (SMC) protein complexes, such as condensin and cohesin, and their regulators, including CTCF, in a highly dynamic process known as loop extrusion. Genetic loss of extrusion factors is lethal, complicating their study. Inducible protein degradation technologies enable the depletion of loop extrusion factors within hours, leading to the rapid reconfiguration of chromatin folding. Here, we review how these technologies have changed our understanding of genome organization, upsetting long-held beliefs on its role in transcription. Finally, we examine recent models that attempt to reconcile observations after chronic versus acute perturbations, and discuss future developments in this rapidly developing field of research.

TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei.

ABSTRACTTwenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium nuclear pore complex (NPC) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here, we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the P. berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry, we identified 4 known nucleoporins (Nups) (138, 205, 221, and the bait 313), and verify interaction with the putative phenylalanine-glycine (FG) Nup637; we assigned 5 proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by green fluorescent protein (GFP) tagging. Based on gene deletion attempts, all new Nups — Nup176, 269, 335, 390, and 434 — are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit 2 incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC (Structural Maintenance Of Chromosomes 4). The protocols developed here showcase the power of proximity labeling for elucidating protein complex composition and annotation of taxonomically restricted genes in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position.

A novel structural maintenance of chromosomes (SMC)-related protein family specific to Archaea.

The ATPases belonging to the structural maintenance of chromosomes (SMC) superfamily are involved in the maintenance of chromosome organization and dynamics, as well as DNA repair. The major proteins in this superfamily recognized to date are either conserved among the three domains of Life (i.e., SMC and Rad50) or specific to Bacteria (i.e., RecF, RecN, and MukB). In Archaea, no protein related to SMC (SMC-related protein) with a broad taxonomic distribution has been reported. Nevertheless, two SMC-related proteins, namely coalescin and Sph, have been identified in crenarchaea Sulfolobus spp. and the euryarchaeon Halobacterium salinarum, respectively, hinting that the diversity of SMC-related proteins has been overlooked in Archaea. In this study, we report a novel SMC-related protein that is distributed among broad archaeal lineages and termed “Archaea-specific SMC-related proteins” or “ASRPs.” We further demonstrate that the ASRP family encloses both coalescin and Sph but the two proteins represent only a tip of the diversity of this family.

KSHV RTA antagonizes SMC5/6 complex-induced viral chromatin compaction by hijacking the ubiquitin-proteasome system.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus with the capacity to establish life-long latent infection. During latent infection, the viral genome persists as a circular episome that associates with cellular histones and exists as a nonintegrated minichromosome in the nucleus of infected cells. Chromatin structure and epigenetic programming are required for the proper control of viral gene expression and stable maintenance of viral DNA. However, there is still limited knowledge regarding how the host regulates the chromatin structure and maintenance of episomal DNA. Here, we found that the cellular protein structural maintenance of chromosome (SMC) complex SMC5/6 recognizes and associates with the KSHV genome to inhibit its replication. The SMC5/6 complex can bind to the KSHV genome and suppress KSHV gene transcription by condensing the viral chromatin and creating a repressive chromatin structure. Correspondingly, KSHV employs an antagonistic strategy by utilizing the viral protein RTA to degrade the SMC5/6 complex and antagonize the inhibitory effect of this complex on viral gene transcription. Interestingly, this antagonistic mechanism of RTA is evolutionarily conserved among γ-herpesviruses. Our work suggests that the SMC5/6 complex is a new host factor that restricts KSHV replication.

KSHV Topologically Associating Domains in Latent and Reactivated Viral Chromatin.

ABSTRACTEukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 bp resolution and constructed a 3D KSHV genomic structural model with 2 kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by structural maintenance of chromosomes (SMC1) cohesin complex and CCCTC-binding factor (CTCF), and the KSHV transactivator was recruited to those sites during reactivation. Triggering KSHV gene expression decreased prewired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible noncoding RNA regions moved toward the inner space of the structure, resembling the configuration of a “bird cage” during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help facilitate viral gene transcription.IMPORTANCE The 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi’s sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibited by the host cell genome, essentially behaving as a mini human chromosome. Thus, we hypothesized that the KSHV genome may be organized in a similar manner. In this report, we describe the domain structure of the latent and lytic KSHV genome at 500 bp resolution and present a 3D genomic structural model for KSHV under each condition. These results add new insights into the complex regulation of the viral life cycle.

Characterization of TRIM16, a member of the fish-specific finTRIM family, in olive flounder Paralichthys olivaceus

Tripartite motif-containing (TRIM) proteins are conserved throughout the metazoan kingdom, and the TRIM subset finTRIM is highly diversified in fish. We isolated TRIM16 cDNA, a member of the finTRIM family, from the olive flounder Paralichthys olivaceus (PoTRIM16). PoTRIM16 contained a 1,725-bp coding sequence encoding a 574-amino acid polypeptide, which in turn contained a really interesting new gene (RING) finger domain, B-box-type zinc finger (B-BOX), nuclease SbcCD subunit C (SbcC), structural maintenance of chromosome (SMC prok B), and stonustoxin (SNTX) subunit alpha (SPRY-PRY-SNTX). Multiple alignment of related sequences revealed that PoTRIM16 showed 86.63–97.40% identity with fish orthologues, and a phylogenetic tree was constructed of vertebrates. PoTRIM16 mRNA was detected in all tissues examined; levels were highest in the eye and ovary. PoTRIM16 mRNA expression was investigated during early development. Under VHSV infection, PoTRIM16 mRNA was downregulated in the liver of P. olivaceus. This is the first study to characterize fish-specific finTRIM in P. olivaceus, which may play a role in the immune response against virus infection.

Condensin-driven loop extrusion on supercoiled DNA.

Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, for example, the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping are stimulated by positively supercoiled DNA, and condensin preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.

A diRNA-protein scaffold module mediates SMC5/6 recruitment in plant DNA repair.

In eukaryotes, the STRUCTURAL MAINTENANCE OF CHROMOSOME 5/6 (SMC5/6) complex is critical to maintaining chromosomal structures around double-strand breaks (DSBs) in DNA damage repair. However, the recruitment mechanism of this conserved complex at DSBs remains unclear. In this study, using Arabidopsis thaliana as a model, we found that SMC5/6 localization at DSBs is dependent on the protein scaffold containing INVOLVED IN DE NOVO 2 (IDN2), CELL DIVISION CYCLE 5 (CDC5), and ALTERATION/DEFICIENCY IN ACTIVATION 2B (ADA2b), whose recruitment is further mediated by DNA-damage-induced RNAs (diRNAs) generated from DNA regions around DSBs. The physical interactions of protein components including SMC5-ADA2b, ADA2b-CDC5, and CDC5-IDN2 result in formation of the protein scaffold. Further analysis indicated that the DSB localization of IDN2 requires its RNA-binding activity and ARGONAUTE 2 (AGO2), indicating a role for the AGO2-diRNA complex in this process. Given that most of the components in the scaffold are conserved, the mechanism presented here, which connects SMC5/6 recruitment and small RNAs, will improve our understanding of DNA repair mechanisms in eukaryotes.

Loss of NSE-4 Perturbs Genome Stability and DNA Repair in Caenorhabditis elegans.

The Structural Maintenance of Chromosomes (SMC) complex plays an important role in maintaining chromosome integrity, in which the SMC5/6 complex occupies a central position by facilitating mitotic and meiotic processes as well as DNA repair. NSE-4 Kleisin is critical for both the organization and function of the SMC5/6 complex, bridging NSE1 and NSE3 (MAGE related) with the head domains of the SMC5 and SMC6 proteins. Despite the conservation in protein sequence, no functional relevance of the NSE-4 homologous protein (NSE-4) in Caenorhabditis elegans has been reported. Here, we demonstrated the essential role of C. elegans NSE-4 in genome maintenance and DNA repair. Our results showed that NSE-4 is essential for the maintenance of chromosomal structure and repair of a range of chemically induced DNA damage. Furthermore, NSE-4 is involved in inter-sister repair during meiosis. NSE-4 localizes on the chromosome and is indispensable for the localization of NSE-1. Collectively, our data from this study provide further insight into the evolutionary conservation and diversification of NSE-4 function in the SMC-5/6 complex.

DNA sliding and loop formation by E. coli SMC complex: MukBEF

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

Abstract 2956: Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma

Abstract Background Multiple myeloma (MM) is an incurable malignancy characterized by complex heterogeneous cytogenetic abnormalities. Defined as large clusters of cis-acting enhancers, super-enhancers (SEs) could promote oncogenic transcription to which cancer cells highly addicted. Structural maintenance of chromosome 4 (SMC4) is a core subunit of condensin complexes, which contributes to chromosome condensation and segregation. Here, we identified SMC4 as a novel SE-associated oncogene of myeloma cells, whose functional roles in MM remain largely elusive. Methods We performed H3K27ac ChIP-seq and RNA-seq on primary MM patient samples, MM cell lines, normal CD138+ plasma cell and lymphoma cell lines as the controls, then the ROSE analysis was used to annotate SEs and their associated genes. THZ1 as a transcriptional CDK7 inhibitor was used to further filter THZ1-responsive genes. Combined analysis of THZ1-sensitive and SE-associated gene uncovered a number of promising MM oncogenes as SMC4. The public data mining, RNA interference, CRISPR/Cas9 gene editing system, overexpression and a variety of cellular functional assays were performed to determine the oncogenic effects of SMC4 on MM malignant. Transcriptome analysis with SMC4 silencing and overexpression, and the underlying molecular mechanism of SMC4 oncogenic role in MM are ongoing. Results We have demonstrated SMC4 as one of the SE-associated genes specific to MM patient samples and cell lines. SMC4 was related to myelomagenesis with increased expression from MGUS, SMM to MM cases, and overexpression of SMC4 predicted poor overall survival of MM patients, suggesting its clinical relevance. SMC4 silencing in MM cells suppressed cell proliferation and lead to cell apoptosis. Repression of the SMC4-SE region also led to impairment of cell viability and cell growth, consistent with an oncogenic function. Conclusion In summary, our integrative approached by combing H3H27ac ChIP-seq, RNA-seq and THZ1-sensitive transcripts identified the landscape of SEs and novel oncogenic transcripts as SMC4 in MM. Mapping these acquired SEs and their associated genes may provide novel insight into understanding MM biology, and SMC4 may serve as novel therapeutic targets in MM. Citation Format: Yunlu Jia, Jianbiao Zhou, Tze-King Tan, Tae-Hoon Chung, Yongxia Chen, Enfan Zhang, Zhen Cai, Takaomi Sanda, Wee-Joo Chng. Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2956.

A hold-and-feed mechanism drives directional DNA loop extrusion by condensin.

Structural maintenance of chromosomes (SMC) protein complexes structure genomes by extruding DNA loops, but the molecular mechanism that underlies their activity has remained unknown. We show that the active condensin complex entraps the bases of a DNA loop transiently in two separate chambers. Single-molecule imaging and cryo-electron microscopy suggest a putative power-stroke movement at the first chamber that feeds DNA into the SMC-kleisin ring upon adenosine triphosphate binding, whereas the second chamber holds on upstream of the same DNA double helix. Unlocking the strict separation of "motor" and "anchor" chambers turns condensin from a one-sided into a bidirectional DNA loop extruder. We conclude that the orientation of two topologically bound DNA segments during the SMC reaction cycle determines the directionality of DNA loop extrusion.

Cryo-EM structure of DNA-bound Smc5/6 reveals DNA clamping enabled by multi-subunit conformational changes

Structural maintenance of chromosomes (SMC) complexes are essential for chromatin organization and functions throughout the cell cycle. The cohesin and condensin SMCs fold and tether DNA, while Smc5/6 directly promotes DNA replication and repair. The functions of SMCs rely on their abilities to engage DNA, but how Smc5/6 binds and translocates on DNA remains largely unknown. Here, we present a 3.8 Å cryogenic electron microscopy (cryo-EM) structure of DNA-bound Saccharomyces cerevisiae Smc5/6 complex containing five of its core subunits, including Smc5, Smc6, and the Nse1-3-4 subcomplex. Intricate interactions among these subunits support the formation of a clamp that encircles the DNA double helix. The positively charged inner surface of the clamp contacts DNA in a nonsequence-specific manner involving numerous DNA binding residues from four subunits. The DNA duplex is held up by Smc5 and 6 head regions and positioned between their coiled-coil arm regions, reflecting an engaged-head and open-arm configuration. The Nse3 subunit secures the DNA from above, while the hook-shaped Nse4 kleisin forms a scaffold connecting DNA and all other subunits. The Smc5/6 DNA clamp shares similarities with DNA-clamps formed by other SMCs but also exhibits differences that reflect its unique functions. Mapping cross-linking mass spectrometry data derived from DNA-free Smc5/6 to the DNA-bound Smc5/6 structure identifies multi-subunit conformational changes that enable DNA capture. Finally, mutational data from cells reveal distinct DNA binding contributions from each subunit to Smc5/6 chromatin association and cell fitness. In summary, our integrative study illuminates how a unique SMC complex engages DNA in supporting genome regulation.

Multiple Roles of SMC5/6 Complex during Plant Sexual Reproduction.

Chromatin-based processes are essential for cellular functions. Structural maintenance of chromosomes (SMCs) are evolutionarily conserved molecular machines that organize chromosomes throughout the cell cycle, mediate chromosome compaction, promote DNA repair, or control sister chromatid attachment. The SMC5/6 complex is known for its pivotal role during the maintenance of genome stability. However, a dozen recent plant studies expanded the repertoire of SMC5/6 complex functions to the entire plant sexual reproductive phase. The SMC5/6 complex is essential in meiosis, where its activity must be precisely regulated to allow for normal meiocyte development. Initially, it is attenuated by the recombinase RAD51 to allow for efficient strand invasion by the meiosis-specific recombinase DMC1. At later stages, it is essential for the normal ratio of interfering and non-interfering crossovers, detoxifying aberrant joint molecules, preventing chromosome fragmentation, and ensuring normal chromosome/sister chromatid segregation. The latter meiotic defects lead to the production of diploid male gametes in Arabidopsis SMC5/6 complex mutants, increased seed abortion, and production of triploid offspring. The SMC5/6 complex is directly involved in controlling normal embryo and endosperm cell divisions, and pioneer studies show that the SMC5/6 complex is also important for seed development and normal plant growth in cereals.

Evolutionary diversification of the autophagy-related ubiquitin-like conjugation systems

ABSTRACT Two autophagy-related (ATG) ubiquitin-like conjugation systems, the ATG12 and ATG8 systems, play important roles in macroautophagy. While multiple duplications and losses of the ATG conjugation system proteins are found in different lineages, the extent to which the underlying systems diversified across eukaryotes is not fully understood. Here, in order to understand the evolution of the ATG conjugation systems, we constructed a transcriptome database consisting of 94 eukaryotic species covering major eukaryotic clades and systematically identified ATG conjugation system components. Both ATG10 and the C-terminal glycine of ATG12 are essential for the canonical ubiquitin-like conjugation of ATG12 and ATG5. However, loss of ATG10 or the C-terminal glycine of ATG12 occurred at least 16 times in a wide range of lineages, suggesting that possible covalent-to-non-covalent transition is not limited to the species that we previously reported such as Alveolata and some yeast species. Some species have only the ATG8 system (with conjugation enzymes) or only ATG8 (without conjugation enzymes). More than 10 species have ATG8 homologs without the conserved C-terminal glycine, and Tetrahymena has an ATG8 homolog with a predicted transmembrane domain, which may be able to anchor to the membrane independent of the ATG conjugation systems. We discuss the possibility that the ancestor of the ATG12 and ATG8 systems is more similar to ATG8. Overall, our study offers a whole picture of the evolution and diversity of the ATG conjugation systems among eukaryotes, and provides evidence that functional diversifications of the systems are more common than previously thought. Abbreviations: APEAR: ATG8–PE association region; ATG: autophagy-related; LIR: LC3-interacting region; NEDD8: neural precursor cell expressed, developmentally down-regulated gene 8; PE: phosphatidylethanolamine; SAMP: small archaeal modifier protein; SAR: Stramenopiles, Alveolata, and Rhizaria; SMC: structural maintenance of chromosomes; SUMO: small ubiquitin like modifier; TACK: Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota; UBA: ubiquitin like modifier activating enzyme; UFM: ubiquitin fold modifier; URM: ubiquitin related modifier.

Single amino acid substitutions in hydrophobic cores at a head-coiled coil junction region of cohesin facilitate its release of DNA during anaphase.

Cohesin holds sister chromatids together and is cleaved by separase/Cut1 to release DNA during the transition from mitotic metaphase to anaphase. The cohesin complex consists of heterodimeric structural maintenance of chromosomes (SMC) subunits (Psm1 and Psm3), which possess a head and a hinge, separated by long coiled coils. Non-SMC subunits (Rad21, Psc3 and Mis4) bind to the SMC heads. Kleisin/Rad21's N-terminal domain (Rad21-NTD) interacts with Psm3's head-coiled coil junction (Psm3-HCJ). Spontaneous mutations that rescued the cleavage defects in temperature-sensitive (ts) separase mutants were identified in the interaction interface, but the underlying mechanism is yet to be understood. Here, we performed site-directed random mutagenesis to introduce single amino acid substitutions in Psm3-HCJ and Rad21-NTD, and then identified 300 mutations that rescued the cohesin-releasing defects in a separase ts mutant. Mutational analysis indicated that the amino acids involved in hydrophobic cores (which may be in close contact) in Psm3-HCJ and Rad21-NTD are hotspots, since 80 mutations (approx. 27%) were mapped in these locations. Properties of these substitutions indicate that they destabilize the interaction between the Psm3 head and Rad21-NTD. Thus, they may facilitate sister chromatid separation in a cleavage-independent way through cohesin structural re-arrangement.

Clamping of DNA shuts the condensin neck gate

SignificanceDNA needs to be compacted to fit into nuclei and during cell division, when dense chromatids are formed for their mechanical segregation, a process that depends on the protein complex condensin. It forms and enlarges loops in DNA through loop extrusion. Our work resolves the atomic structure of a DNA-bound state of condensin in which ATP has not been hydrolyzed. The DNA is clamped within a compartment that has been reported previously in other structural maintenance of chromosomes (SMC) complexes, including Rad50, cohesin, and MukBEF. With the caveat of important differences, it means that all SMC complexes cycle through at least some similar states and undergo similar conformational changes in their head modules, while hydrolyzing ATP and translocating DNA.

Differential gene expression in tall fescue tissues in response to water deficit.

Tall fescue (Festuca arundinacea Schreb.) is a popular pasture and turf grass particularly known for drought resistance, allowing for its persistence in locations that are unfavorable for other cool-season grasses. Also, its seed-borne fungal symbiont (endophyte) Epichloë coenophiala, which resides in the crown and pseudostem, can be a contributing factor in its drought tolerance. Because it contains the apical meristems, crown survival under drought stress is critical to plant survival as well as the endophyte. In this study, we subjected tall fescue plants with their endophyte to water-deficit stress or, as controls with normal watering, then compared plant transcriptome responses in four vegetative tissues: leaf blades, pseudostem, crown, and roots. A transcript was designated a differentially expressed gene (DEG) if it exhibited at least a twofold expression difference between stress and control samples with an adjusted p value of .001. Pathway analysis of the DEGs across all tissue types included photosynthesis, carbohydrate metabolism, phytohormone biosynthesis and signaling, cellular organization, and a transcriptional regulation. While no specific pathway was observed to be differentially expressed in the crown, genes encoding auxin response factors, nuclear pore anchors, structural maintenance of chromosomes, and class XI myosin proteins were more highly differentially expressed in crown than in the other vegetative tissues, suggesting that regulation in expression of these genes in the crown may aid in survival of the meristems in the crown.

Comprehensive Analysis of SMC Gene Family Prognostic Value and Immune Infiltration in Patients With Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a malignant tumor with high morbidity and mortality worldwide. Members from the structural maintenance of chromosomes (SMC) gene family function as oncogenes in various tumor types, but their roles in PAAD have not been elucidated. In this study, we aimed to explore the role of the SMC family in tumor progression and cancer immune infiltration in PAAD using integrative bioinformatic analyses. The results showed that the SMC 1A, 2, 3, 4, and 6 were overexpressed in PAAD tissues; of these, SMC 1A, 4, 5, and 6 could be potential prognostic biomarkers for PAAD. The expression of SMC genes was found to be strongly associated with immune cell infiltration. According to the infiltrative status of various immune cells, the mRNA expression of SMC genes in PAAD was associated with the overall and recurrence-free survival of patients. In conclusion, the SMC gene family is associated with PAAD and may be involved in tumorigenesis and cancer-immune interactions; thus, members from this gene family may serve as promising prognostic and therapeutic biomarkers of PAAD.

Identification of monomorphic and polymorphic genes associated with recessive fertility defects in Holstein cows reared in Kazakhstan

Haplotypes of candidate genes namely: apoptotic protease activating factor 1 (APAF1 p.Q579X or HH1), glycinamide ribonucleotide formyltransferase (GART or HH4), structural maintenance of chromosomes 2 (SMC2 or HH3), and haplotype cholesterol deficiency (HCD) genes associated with recessive fertility defects (loss of fertility) were investigated in imported Canadian Holstein cows reared at “Medeu Commerce” LLP breeding farm in Kazakhstan. The genotypic profiling of the APAF1/HH1, GART/HH4 fertility haplotype carriers was carried out by PCR-RFLP methods using BstC8I and Tru9I and MseI, while the genotypic profiling of the SMC2/HH3, and HCD fertility haplotype carriers was carried out using our own primer designed by internal primer marker methods. The study revealed that the PCR-RFLP diagnostic markers APAF1/HH1 and GART/HH4 for recessive fertility defects were monomorphic in the Canadian Holstein cows investigated. However, the diagnostic markers SMC2/HH3 and HCD fertility haplotype carriers (our own design diagnostic markers) were polymorphic, with frequencies of 3% and 11%, respectively, in the investigated Canadian Holstein cows. The study concluded that genetic monitoring of recessive fertility defects enables the timely identification of carriers of harmful lethal mutations, and control of the fertility haplotype elimination process.