The mouse Sry locus harbors a cryptic exon that is essential for male sex determination

Science ◽  
2020 ◽  
Vol 370 (6512) ◽  
pp. 121-124 ◽  
Author(s):  
Shingo Miyawaki ◽  
Shunsuke Kuroki ◽  
Ryo Maeda ◽  
Naoki Okashita ◽  
Peter Koopman ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Ectopic Expression ◽  
Cryptic Exon ◽  
Male Development ◽  
C Terminus ◽  
Bona Fide ◽  
Male Sex ◽  
Determining Factor ◽  
Single Exon Gene ◽  
Exon Gene

The mammalian sex-determining gene Sry induces male development. Since its discovery 30 years ago, Sry has been believed to be a single-exon gene. Here, we identified a cryptic second exon of mouse Sry and a corresponding two-exon type Sry (Sry-T) transcript. XY mice lacking Sry-T were sex-reversed, and ectopic expression of Sry-T in XX mice induced male development. Sry-T messenger RNA is expressed similarly to that of canonical single-exon type Sry (Sry-S), but SRY-T protein is expressed predominantly because of the absence of a degron in the C terminus of SRY-S. Sry exon2 appears to have evolved recently in mice through acquisition of a retrotransposon-derived coding sequence to replace the degron. Our findings suggest that in nature, SRY-T, not SRY-S, is the bona fide testis-determining factor.

The evolution of mammalian sex chromosomes and the origin of sex determining genes

1995 ◽  
Vol 350 (1333) ◽  
pp. 305-312 ◽  
Keyword(s):  
Sex Determination ◽  
Sex Chromosomes ◽  
Pseudoautosomal Region ◽  
Comparative Gene Mapping ◽  
Male Development ◽  
Male Sex ◽  
Close Relatives ◽  
Determining Factor ◽  
Chromosomal Sex Determination ◽  
Active Genes

Mammals have XX female :XY male chromosomal sex determination in which a small heterochromatic Y controls male development. Only a few active genes have been identified on the Y, including the testis determining factor SRY and candidate spermatogenesis genes. These genes, as well as several pseudogenes, have close relatives on the X, confirming that the Y was originally homologous to the X, but has been progressively degraded. We used comparative gene mapping of sex chromosomes from the three major groups of extant mammals (eutherians, marsupials and monotremes) to deduce how the X and Y evolved from a pair of autosomes, and how SRY assumed control of sex determination. We found that part of the X, and a corresponding region of the Y chromosome, is shared by all mammals and must be very ancient, but part of the X (and Y) was added quite recently. I propose that a small original X and Y were enlarged by cycles of autosomal addition to one partner, recombination onto the other and continuing attrition of the compound Y. This addition-attrition hypothesis predicts that the pseudoautosomal region of the human X is merely a relic of the last addition, and that the gene content of the pseudoautosomal region may well differ in different mammalian lineages. The only genes which remained active on the conserved or added regions of the Y were those, like SRY , that evolved functions in male sex determination and differentiation distinct from the general functions of their X-linked partners. Although the vertebrate gonadogenesis pathway is highly conserved, its control circuitry has probably changed radically and rapidly in evolution.

scholarly journals The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 926
Author(s):  
Maria C. Martins ◽  
Susana F. Fernandes ◽  
Bruno A. Salgueiro ◽  
Jéssica C. Soares ◽  
Célia V. Romão ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Reductase Activity ◽  
Conserved Residues ◽  
Mutant Proteins ◽  
E Coli ◽  
C Terminus ◽  
Bona Fide ◽  
Oxygen Reductase ◽  
Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

scholarly journals Common Spontaneous Sex-Reversed XX males of the Medaka Oryzias latipes

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 245-251 ◽  
Author(s):  
Indrajit Nanda ◽  
Ute Hornung ◽  
Mariko Kondo ◽  
Michael Schmid ◽  
Manfred Schartl
Keyword(s):  
Y Chromosome ◽  
Oryzias Latipes ◽  
Fish Analysis ◽  
Normal Phenotype ◽  
Dmrt1 Gene ◽  
Male Development ◽  
Female Bias ◽  
Male Sex ◽  
Xx Males ◽  
Number Of Males

Abstract In the medaka, a duplicated version of the dmrt1 gene, dmrt1bY, has been identified as a candidate for the master male sex-determining gene on the Y chromosome. By screening several strains of Northern and Southern medaka we identified a considerable number of males with normal phenotype and uncompromised fertility, but lacking dmrt1bY. The frequency of such males was >10% in some strains and zero in others. Analysis for the presence of other Y-linked markers by FISH analysis, PCR, and phenotype indicated that their genotype is XX. Crossing such males with XX females led to a strong female bias in the offspring and also to a reappearance of XX males in the following generations. This indicated that the candidate male sex-determining gene dmrt1bY may not be necessary for male development in every case, but that its function can be taken over by so far unidentified autosomal modifiers.

scholarly journals Application of a Novel Strategy of Engineering Conditional Alleles to a Single Exon Gene, Sox2

PLoS ONE ◽  
2012 ◽  
Vol 7 (9) ◽  
pp. e45768 ◽  
Author(s):  
Nikolaos Mandalos ◽  
Marannia Saridaki ◽  
Jessica Lea Harper ◽  
Anastasia Kotsoni ◽  
Peter Yang ◽  
...  
Keyword(s):  
Novel Strategy ◽  
Single Exon Gene ◽  
Exon Gene

scholarly journals Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor

2018 ◽  
Vol 96 (5) ◽  
pp. 459-470 ◽  
Author(s):  
Xavier Charest-Morin ◽  
Robert Lodge ◽  
François Marceau
Keyword(s):  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Denaturation ◽  
Epifluorescence Microscopy ◽  
Terminal Sequence ◽  
Protein Ligands ◽  
C Terminus ◽  
Bona Fide ◽  
Green Fluorescent

To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3′,5,5′-tetramethylbenzidine (luminescence or colourimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range of 50–600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

scholarly journals Distinct Contributions of Conserved Modules to Runt Transcription Factor Activity

2010 ◽  
Vol 21 (13) ◽  
pp. 2315-2326 ◽  
Author(s):  
Pegine B. Walrad ◽  
Saiyu Hang ◽  
Genevieve S. Joseph ◽  
Julia Salas ◽  
J. Peter Gergen
Keyword(s):  
Transcriptional Activation ◽  
Ectopic Expression ◽  
Developmental Pathways ◽  
Transcription Factor Activity ◽  
Factor Activity ◽  
Runt Domain ◽  
Animal Kingdom ◽  
Axonal Projections ◽  
C Terminus ◽  
Runx Proteins

Runx proteins play vital roles in regulating transcription in numerous developmental pathways throughout the animal kingdom. Two Runx protein hallmarks are the DNA-binding Runt domain and a C-terminal VWRPY motif that mediates interaction with TLE/Gro corepressor proteins. A phylogenetic analysis of Runt, the founding Runx family member, identifies four distinct regions C-terminal to the Runt domain that are conserved in Drosophila and other insects. We used a series of previously described ectopic expression assays to investigate the functions of these different conserved regions in regulating gene expression during embryogenesis and in controlling axonal projections in the developing eye. The results indicate each conserved region is required for a different subset of activities and identify distinct regions that participate in the transcriptional activation and repression of the segmentation gene sloppy-paired-1 (slp1). Interestingly, the C-terminal VWRPY-containing region is not required for repression but instead plays a role in slp1 activation. Genetic experiments indicating that Groucho (Gro) does not participate in slp1 regulation further suggest that Runt's conserved C-terminus interacts with other factors to promote transcriptional activation. These results provide a foundation for further studies on the molecular interactions that contribute to the context-dependent properties of Runx proteins as developmental regulators.

scholarly journals Porcine Haemagglutinating Encephalomyelitis Virus Triggers Neural Autophagy Independent of ULK1

2021 ◽  
Author(s):  
Zi Li ◽  
Feng Gao ◽  
Yungang Lan ◽  
Jiyu Guan ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Time Course ◽  
Ectopic Expression ◽  
Neuroblastoma Cells ◽  
Neurological Dysfunction ◽  
Global Public Health ◽  
Complex Interactions ◽  
Encephalomyelitis Virus ◽  
Bona Fide

Uncoordinated 51-like kinase 1 (ULK1) is a well-characterized initiator of canonical autophagy under basal or pathological conditions. Porcine haemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus (β-CoV), impairs ULK1 kinase but hijacks autophagy to facilitate viral proliferation. However, the machinery of PHEV-induced autophagy initiation upon ULK1 kinase deficiency remains unclear. Here, the time course of PHEV infection showed a significant accumulation of autophagosomes (APs) in nerve cells in vivo and in vitro. Utilizing the ULK1-knockout neuroblastoma cells, we have identified that ULK1 was not essential for productive AP formation induced by PHEV. In vitro phosphorylation studies discovered that mTORC1-regulated ULK1 activation stalls during PHEV infection, whereas the AP biogenesis was controlled by AMPK-driven BECN1 phosphorylation. A lack of BECN1 is sufficient to block LC3 lipidation and disrupt recruitment of the LC3-ATG14 complex. Moreover, BECN1 acts as a bona fide substrate for ULK1-independent neural autophagy, and ectopic expression of BECN1 somewhat enhances PHEV replication. These findings highlight a novel machinery of non-canonical autophagy independent of ULK1 that bypasses the conserved initiation circuit of AMPK-mTORC1-ULK1, providing new insights into the interplay between neurotropic β-CoV and the host. IMPORTANCE The ongoing COVID-19 pandemic alongside the outbreaks of SARS and MERS pose betacoronavirus (β-CoV) as a global public health challenge. Coronaviruses subvert, haijack, or utilize autophagy to promote proliferation, thus exploring the cross-talk between β-CoV and autophagy of great significance in confronting future β-CoV outbreaks. Porcine haemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic β-CoV and invades the central nervous system (CNS) in pigs, but understanding of the pathogenesis for PHEV-induced neurological dysfunction yet limited. Here, we discovered a novel regulatory principle of neural autophagy initiation during PHEV infection, where productive autophagosome (AP) biogenesis bypassing the multifaceted regulation of ULK1 kinase. The PHEV-triggered non-canonical autophagy underscores the complex interactions of virus-host, and will help in the development of therapeutic strategies targeting non-canonical autophagy to treat β-CoV disease.

scholarly journals Plk is a functional homolog of Saccharomyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation structures.

1997 ◽  
Vol 17 (6) ◽  
pp. 3408-3417 ◽  
Author(s):  
K S Lee ◽  
R L Erikson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Specific Activity ◽  
Ectopic Expression ◽  
Sf9 Cells ◽  
Wild Type ◽  
C Terminus ◽  
M Phase

Plk is a mammalian serine/threonine protein kinase whose activity peaks at the onset of M phase. It is closely related to other mammalian kinases, Snk, Fnk, and Prk, as well as to Xenopus laevis Plx1, Drosophila melanogaster polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae Cdc5. The M phase of the cell cycle is a highly coordinated process which insures the equipartition of genetic and cellular materials during cell division. To enable understanding of the function of Plk during M phase progression, various Plk mutants were generated and expressed in Sf9 cells and budding yeast. In vitro kinase assays with Plk immunoprecipitates prepared from Sf9 cells indicate that Glu206 and Thr210 play equally important roles for Plk activity and that replacement of Thr210 with a negatively charged residue elevates Plk specific activity. Ectopic expression of wild-type Plk (Plk WT) complements the cell division defect associated with the cdc5-1 mutation in S. cerevisiae. The degree of complementation correlates closely with the Plk activity measured in vitro, as it is enhanced by a mutationally activated Plk, T210D, but is not observed with the inactive forms K82M, D194N, and D194R. In a CDC5 wild-type background, expression of Plk WT or T210D, but not of inactive forms, induced a sharp accumulation of cells in G1. Consistent with elevated Plk activity, this phenomenon was enhanced by the C-terminally deleted forms WT deltaC and T210D deltaC. Expression of T210D also induced a class of cells with unusually elongated buds which developed multiple septal structures. This was not observed with the C-terminally deleted form T210D deltaC, however. It appears that the C terminus of Plk is not required for the observed cell cycle influence but may be important for polarized cell growth and septal structure formation.

scholarly journals Generating Bona Fide Mammalian Prions with Internal Deletions

2016 ◽  
Vol 90 (15) ◽  
pp. 6963-6975 ◽  
Author(s):  
Carola Munoz-Montesino ◽  
Christina Sizun ◽  
Mohammed Moudjou ◽  
Laetitia Herzog ◽  
Fabienne Reine ◽  
...  
Keyword(s):  
De Novo ◽  
Alpha Helix ◽  
Transmissible Spongiform Encephalopathies ◽  
Specific Information ◽  
Beta Sheet ◽  
Internal Deletion ◽  
C Terminus ◽  
Parkinson’S Diseases ◽  
Bona Fide ◽  
Mammalian Prions

ABSTRACTMammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating andde novoinfectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions.IMPORTANCEPrions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

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