TY  - JOUR
A1  - Rieck, Christoph Paul Kurt
A1  - Geiger, Daniel
A1  - Munkert, Jennifer
A1  - Messerschmidt, Katrin
A1  - Petersen, Jan
A1  - Strasser, Juliane
A1  - Meitinger, Nadine
A1  - Kreis, Wolfgang
T1  - Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae
JF  - Microbiologyopen
N2  - A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5‐3β‐hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ5‐isomerase gene from Comamonas testosteronii, (c) a mutated steroid‐5β‐reductase gene from Arabidopsis thaliana, and (d) a steroid 21‐hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed “CARD II yeast”, was capable of producing 5β‐pregnane‐3β,21‐diol‐20‐one, a central intermediate in 5β‐cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast.
Y1  - 2019
U6  - https://doi.org/10.1002/mbo3.925
SN  - 2045-8827
VL  - 8
IS  - 12
PB  - Wiley
CY  - Hoboken
ER  - 
TY  - JOUR
A1  - Stuckas, Heiko
A1  - Messerschmidt, Katrin
A1  - Putzler, Sascha
A1  - Baumann, Otto
A1  - Schenk, Jörg A.
A1  - Tiedemann, Ralph
A1  - Micheel, Burkhard
T1  - Detection and characterization of gamete-specific molecules in Mytilus edulis using selective antibody production
N2  - The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76%) which explains the cross reactivity of mAb G26- AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals.
Y1  - 2009
UR  - http://www3.interscience.wiley.com/cgi-bin/jhome/37692
U6  - https://doi.org/10.1002/Mrd.20916
SN  - 1040-452X
ER  -