Chromosomal assignment of six muscle-specific genes in cattle

Six genes expressed in skeletal or smooth muscle were assigned to bovine chromosomes using rodent, human or bovine cDNA probes. Myogenic determination factor (MYOD1) was 100% concordant with Bos taurus chromosome (BTA) 15, and myogenin (MYOG) was 95% concordant with BTA 16. Smooth muscle caldesmon (CALD1) and the skeletal muscle chloride channel gene (CLCN1) were 100% concordant with BTA 4. Myogenic factor 5 (MYF5) was 90% concordant with BTA 5; this assignment was confirmed by fluorescence in situ hybridization of a bovine genomic MYF5 probe to BTA 5 band 13 and the homologous band on river buffalo 4q. In some metaphases, specific hybridization signals were also observed on BTA 15 band 23, and the equivalent river buffalo homologue, with the MYF5 genomic probe. Because MYOD1 and MYF5 share both nucleotide and functional homology and because MYOD1 was mapped in somatic cell hybrids to BTA 15, we suggest that MYOD1 may be located at BTA 15 band 23. Herculin/myogenic factor 6 (MYF6) was assigned indirectly to BTA 5 by the hybridization of MYF5 and MYF6 probes to the same Hin dIII fragment in bovine genomic DNA. The assignment of MYF6 to BTA 5 is consistent with the tandem arrangement of MYF5 and MYF6 in human, mouse and chicken, where these tightly linked genes are separated by < 6·5 kb of DNA.     

Allelic Variation in the Porcine MYF5 Gene Detected by PCR–SSCP

The MYF5 gene has been reported to be integral to muscle growth and development, and hence it has been considered as a candidate gene for meat selection programs in pig. To ascertain whether there was variation in the porcine MYF5 gene, we have developed a method of PCR–single-strand conformational polymorphism (PCR–SSCP) analysis. In this study, two coding regions of the MYF5 gene were investigated. Four unique SSCP patterns were detected in exon 1 and three patterns were identified in exon 3. Two SNPs detected in exon 1 led to a non-synonymous alanine/proline substitution. A nucleotide change in exon 3 did not affect the amino acid sequence. Five extended haplotypes were observed across the two regions. The variation detected in this study might underpin the development of gene markers for improved muscle growth in pig breeding.     

<Emphasis Type="Italic">smyd1</Emphasis> and <Emphasis Type="Italic">smyd2</Emphasis> are expressed in muscle tissue in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Epigenetic modifications of histone play important roles for regulation of cell activity, such as cell division, cell death, and cell differentiation. A SET domain consisting of about 130 amino acids has lysine methyltransferase activity in the presence of the cosubstrate S-adenosyl-methionine. More than 60 SET domain-containing proteins have been predicted in various organisms. One of them, the SMYD family genes which contain a SET domain and a zinc-finger MYND domain are reported to regulate cell cycle and muscle formation. Here we examined the expression and function of smyd1 and 2 in Xenopus. smyd1 and 2 were expressed in various muscle tissues. While smyd1 expression was observed mainly in cardiac muscle and skeletal muscle, smyd2 expression was done abundantly in skeletal muscle and face region. Moreover, by loss-of-function experiments using antisense morpholino oligonucleotides, it was suggested that smyd1 and 2 related to muscle cells differentiation.     

Ethylene signaling is critical for synergid cell functional specification and pollen tube attraction

ETHYLENE INSENSITIVE 3 (EIN3) is a key regulator of ethylene signaling, and EIN3‐BINDING F‐BOX1 (EBF1) and EBF2 are responsible for EIN3 degradation. Previous reports have shown that the ebf1 ebf2 double homozygous mutant cannot be identified. In this study, the genetic analysis revealed that the ebf1 ebf2 female gametophyte is defective. The pollination experiment showed that ebf1 ebf2 ovules failed to attract pollen tubes. In female gametophyte/ovule, the synergid cell is responsible for pollen tube attraction. Observation of the pEIN3::EIN3‐GFP transgenic lines showed that EIN3 signal was over‐accumulated at the micropylar end of ebf1 ebf2 female gametophyte. The overexpression of stabilized EIN3 in synergid cell led to the defect of pollen tube guidance. These results suggested that the over‐accumulated EIN3 in ebf1 ebf2 synergid cell blocks its pollen tube attraction which leads to the failure of ebf1 ebf2 homozygous plant. We identified that EIN3 directly activated the expression of a sugar transporter, SENESCENCE‐ASSOCIATED GENE29 (SAG29/SWEET15). Overexpression of SAG29 in synergid cells blocked pollen tube attraction, suggesting that SAG29 might play a role in ethylene signaling to repel pollen tube entry. Taken together, our study reveals that strict control of ethylene signaling is critical for the synergid cell function during plant reproduction.     

A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.     

Activin A signaling directly activates Xenopus winged helix factors XFD-4/4’, the orthologues to mammalian MFH-1

We investigated the Xenopus winged helix gene XFD-4, its cDNA, and a pseudoallelic cDNA, termed XFD-4’, representing Xenopus orthologues to chicken CWH-2 and mammalian MFH-1. XFD-4/4’ genes are activated after midblastula transition in dorsolateral mesoderm but not within the dorsal lip. Later, expression is found in two segmented lines of cells bordering the somites, in head mesenchyme, in ventral abdominal muscle, and in the tail tip. Smad2 RNA injection leads to ectopic expression of XFD-4’. Since activation is also observed in activin A treated animal cap explants in the presence of cycloheximide, XFD-4/4’ genes represent direct targets of activin signaling. Note that the future nomenclature for XFD-4 will be FoxC2a and for XFD-4’ will be FoxC2b (Fox Nomenclature Committee). Received: 1 September 1999 / Accepted: 16 December 1999     

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.     

Myogenin is in an evolutionarily conserved linkage group on human chromosome 1q31-q41 and unlinked to other mapped muscle regulatory factor genes

Myogenin is a member of a family of muscle-specific regulatory factors which includes MyoD1, Myf-5, and Myf-6 (also called MRF4 and herculin). Extensive regions of sequence homology in genes for these three factors suggest duplication events associated with their evolution. In the present study, the chromosomal location of the myogenin gene in humans (MYOG), mice (Myog), and Chinese hamsters (MYOG) was determined using in situ hybridization to human metaphase chromosomes as well as segregation analysis among interspecific somatic cell hybrid panels and interspecific backcrossed mice. We localize the gene encoding myogenin to human chromosome 1q31-q41 within a linkage group homologous with a region on mouse chromosome 1 and Chinese hamster chromosome 5. The results verify the nonlinkage of MYOG to MYOD1, MYF5, and MYF6 genes and indicate that events associated with the duplication of MYOG with respect to MYOD1, MYF5, or MYF6 loci were not chromosome-wide.     

Identification of key candidate biomarkers for severe influenza infection by integrated bioinformatical analysis and initial clinical validation

One of the key barriers for early identification and intervention of severe influenza cases is a lack of reliable immunologic indicators. In this study, we utilized differentially expressed genes screening incorporating weighted gene co-expression network analysis in one eligible influenza GEO data set ( GSE111368 ) to identify hub genes associated with clinical severity. A total of 10 genes (PBI, MMP8, TCN1, RETN, OLFM4, ELANE, LTF, LCN2, DEFA4 and HP) were identified. Gene set enrichment analysis (GSEA) for single hub gene revealed that these genes had a close association with antimicrobial response and neutrophils activity. To further evaluate these genes' ability for diagnosis/prognosis of disease developments, we adopted double validation with (a) another new independent data set ( GSE101702 ); and (b) plasma samples collected from hospitalized influenza patients. We found that 10 hub genes presented highly correlation with disease severity. In particular, BPI and MMP8 encoding proteins in plasma achieved higher expression in severe and dead cases, which indicated an adverse disease development and suggested a frustrating prognosis. These findings provide new insight into severe influenza pathogenesis and identify two significant candidate genes that were superior to the conventional clinical indicators. These candidate genes or encoding proteins could be biomarker for clinical diagnosis and therapeutic targets for severe influenza infection.