Low temperature is among the important factors affecting the distribution, survival, growth, and physiology of aquatic animals. In this study, coordinated transcriptomic responses to 10 °C acute cold stress were investigated in the gills, hearts, livers, and spleens of Japanese flounder (Paralichthys olivaceus), an important aquaculture species in east Asia. Histological examination suggested different levels of injury among P. olivaceus tissues after cold shock, mainly in the gills and livers. Based on transcriptome and weighted gene coexpression network analysis, 10 tissue-specific cold responsive modules (CRMs) were identified, revealing a cascade of cellular responses to cold stress. Specifically, five upregulated CRMs were enriched with induced differentially expressed genes (DEGs), mainly corresponding to the functions of "extracellular matrix", "cytoskeleton", and "oxidoreductase activity", indicating the induced cellular response to cold shock. The "cell cycle/division" and "DNA complex" functions were enriched in the downregulated CRMs for all four tissues, which comprised inhibited DEGs, suggesting that even with tissue-specific responses, cold shock may induce severely disrupted cellular functions in all tissues, reducing aquaculture productivity. Therefore, our results revealed the tissue-specific regulation of the cellular response to low-temperature stress, which warrants further investigation and provides more comprehensive insights for the conservation and cultivation of P. olivaceus in cold water.

Minichromosome maintenance (MCM) complexes have been identified as the primary replicative helicases responsible for unwinding DNA for genome replication. The focus of this chapter is to discuss the current structural and functional understanding of MCMs and their role at origins of replication, which are based mostly on the studies of MCM proteins and MCM complexes from archaeal genomes.

Minichromosome maintenance protein (MCM) is composed of six structurally related subunits (MCM2-7) and is essential for eukaryotic DNA replication initiation and early stage of elongation process. Recently human and Xenopus MCM8 was identified as a novel member of MCM protein. Here we characterized MCM8 orthologous genes by using bioinformatics. Human MCM8 showed approximately 90%, 90%, 93%, and 79% total-amino acid identity with mouse, rat, dog, and chicken MCM8, respectively. Human, mouse, rat, dog, and chicken MCM8 gene, consisting of 19, 18, 17, 18, and 18 exons, was mapped to 20p12.3-13, 2F3, 3q36, 24, and 3, respectively. We identified transcription factor E2F-binding motifs in the vicinity of the transcription start site among MCM8 orthologous genes. The mammalian but not chicken E2F-binding motif was accompanied by NF-Y binding motif. MCM8 mRNA was upregulated by E2E1 in human culture cells. Chromatin immunoprecipitation (ChIP) demonstrated the direct association of E2F1 and NF-Y with human MCM8 promoter. The promoter activities of human, rat, and chicken MCM8 were demonstrated to be E2F1-dependent. Analysis of human MCM8 promoter constructs showed that an E2F-binding motif in the vicinity of the transcription initiation site is necessary for the transcriptional activation. We also showed that the transcription of human MCM8 is activated by transcription factors E2F1-4, but not by factors E2F5-8.

The minichromosome maintenance (MCM) proteins are essential for DNA replication initiation and elongation in eukaryotes. In mammalian cells, MCM2-MCM7 complexes are believed to unwind DNA during chromosomal DNA replication. Here we identified a novel MCM family gene, MCM9, by using bioinformatics. Human, mouse, and rat MCM9 showed approximately 90-91% total-amino acid identity. MCM9 showed 24-31% total-amino acid identity with MCM2-MCM8 protein. Phylogenetic analysis on MCM family members revealed that MCM9 was most closely related to MCM8. Human, mouse, and rat MCM9 gene, consisting of 7, 8, and 7 exons, was mapped to 6q22.1-22.33, 10B3, and 20q11, respectively. We identified transcription factor E2F-binding motifs in the vicinity of the transcription start site among human, mouse, and rat MCM9 gene. MCM9 mRNA was upregulated by transcription factor E2E1 and serum stimulation in NIH3T3 cells.

The minichromosome maintenance (MCM) 2-7 complex is a putative DNA helicase complex that facilitates the initiation of DNA replication. Here, we generated a cell line MCM7(+/-)/MCM7-FLAG, in which one allele of MCM7 is mutated whereas a tetracycline-repressible promoter could manipulate the expression of exogenous MCM7 protein. Overexpressed MCM7 protein supports efficient DNA replication of Epstein-Barr virus oriP and rapid formation of tumors in nude mice without altering the activity of cellular DNA replication. This system provides a unique setting for studying the function of MCM7 and for screening for potential therapeutics for malignant tumors.

Previous work with yeast cells and with Xenopus egg extracts had shown that eukaryotic pre-replication complexes assemble on chromatin in a step-wise manner whereby specific loading factors promote the recruitment of essential Mcm proteins at pre-bound origin recognition complexes (ORC with proteins Orc1p-Orc6p). While the order of assembly--Mcm binding follows ORC binding--seems to be conserved in cycling mammalian cells in culture, it has not been determined whether mammalian Mcm proteins associate with ORC-bearing chromatin sites. We have used a chromatin immunoprecipitation approach to investigate the site of Mcm binding in a genomic region that has previously been shown to contain an ORC-binding site and an origin of replication. Using chromatin from HeLa cells in G1 phase, antibodies against Orc2p as well as antibodies against Mcm proteins specifically immunoprecipitate chromatin enriched for a DNA region that includes a replication origin. However, with chromatin from cells in S phase, only Orc2p-specific antibodies immunoprecipitate the origin-containing DNA region while Mcm-specific antibodies immunoprecipitate chromatin with DNA from all parts of the genomic region investigated. Thus, human Mcm proteins first assemble at or adjacent to bound ORC and move to other sites during genome replication.

The six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AAA(+) ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits.