This featurean enlarged male geneis found in many species with DUI; the COX2 protein can be up to 420 amino acids very long. male-transmitted mtDNA in gene (Minclude long 3 extensions or in-frame insertions [15C21]. For example, freshwater mussels (order Unionoida) have a fast-evolving 3 extension of variable size (144C681 bp) in their Mgene, but still display a pattern of purifying selection with this additional sequence [15C17,19,21]. This extension is definitely transcribed and translated, and its c-terminus tail has been localized in the mitochondrial surface of sperm mitochondria, leading to the hypothesis that it could serve as a tag to facilitate their sex-specific fate in bivalve embryos [17,22,23]. However, more work is needed to understand the practical importance of these modifications in the MCOX2 protein of bivalves with DUI. The longest in-frame insertion (4827 bp) in the Mgene of a DUI bivalve has recently been reported in (order Cardiida) [20], meaning that this gene could be expressed as a single polypeptide drastically larger (1892 amino acids) than the standard one (approx. 230 aa) or the enlarged GSK2838232A one found in some male mitogenomes of additional DUI varieties (up to 420 aa) [15C17,19C21]. However, this possibility has not been evaluated yet. Here, we used a combination of RT-PCR, immunoassays and comparative genetics to better characterize the Rabbit Polyclonal to OR10J3 development and functionality of this newly found out Minsertion in and confirmed the living of the largest mtDNA-encoded protein reported inside a metazoan so far. 2. ?Material and methods Adult specimens of were collected in May 2013 from Concarneau (France; 47.8728 N, 3.9207 W), and June 2018 and 2019 from Fouras-les-Bains (France; 45.9838 N, 1.0931 W). Individuals were dissected by trimming adductor muscle tissue and sampling a fragment of the mantle. Gonads were gently nicked having a scalpel and gametes were sampled having a P200 pipette and GSK2838232A inspected under the microscope for sexing. Female and male gametes, adult gonads and somatic cells were maintained in 95% ethanol or flash-frozen in liquid nitrogen and sent to the Universit de Montral on dry ice to be stored at ?80C. Total genomic DNA was extracted from gonads and somatic cells with the Qiagen DNeasy Blood & Tissue Kit (QIAGEN 69506). RNA was extracted from gametes and somatic cells with the (R1 and R2) and one region of F(F mtDNA) were PCR-amplified using DNA from 23 males and 18 females (number 1insertion. (genes and the areas targeted by PCR and RT-PCR (R1C3: reddish, blue and orange boxes, respectively; Finsertion is definitely plain. The position of the epitope specific to MCOX2 is definitely demonstrated by an antibody. (and two regions of M(R1 and R3) were PCR-amplified from gamete and somatic cDNA (two-step RT-PCR) (number 1gene, a polyclonal antibody against the synthetic epitope GSK2838232A CDKYKVFPHWE specific to MCOX2 was produced in rabbits and affinity-purified (MediMabs, Montral, Canada). Protein extracts from male and female gametes and somatic cells were supplemented with loading buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% b-mercaptoethanol and 0.02% bromophenol blue) then heated for 5 min at 95C before being loaded into a denaturing 4C20% polyacrylamide gel. Samples migrated for 18 h at 50 V in migration buffer (Tris 25 mM, glycine 192 mM and SDS 0.1%) and were transferred about nitrocellulose membranes at 1000 mA for 1 h 30 min in transfer buffer (glycine 1.5%, Tris base 0.3%,.