Professor Alexander Zawadzki of Lvov university – Gregor Mendel’s mentor and inspirer

It is generally agreed that Johann Gregor Mendel (1822–1884) is the undisputed father of genetics, the study of heredity that is fundamental to our understanding of all living things. However, as a geneticist and engineer, strongly influenced by my teachers in Lvov and Gdansk, in Copenhagen and in Cold Spring Harbor (NY), I have always wondered how it was possible for Mendel to be such an independent thinker while not having a mentor who could have guided his creative and analytical thinking. Was there anybody in Mendel’s life who could have played this role? At the very least, did he get any opportunity to discuss his experimental designs with some colleague or to receive any help from a mentor or a friend in analyzing his results? The purpose in preparing this essay is to bring to light a long neglected story of how an «obscure Polish Professor from Lvov University», Alexander Zawadzki, played a critical role in helping an «obscure Austrian monk», Gregor Mendel, to create the discipline of genetics.Загальновизнано, що Іоганн Грегор Мендель є фундатором генетики – науки, яка вивчає спадковість, що є основою для розуміння всіх життєвих процесів. Однак як генетик, що перебував під впливом моїх вчителів у Львові і Гданьську, Копенгагені і Коулд Спрінг Харборі (Нью-Йорк), я завжди дивувався, як вдалося Менделю бути таким незалежним мислителем за відсутності у нього наставника, який міг би спрямувати його відкриття та аналітичний розум? Чи був хтось у житті Менделя, хто займав таке положення? У крайньому разі, чи була у ньго можливість обговорювати свої експерименти з кимось із колег та одержувати допомогу від керівника або друга в аналізі отриманих результатів? У представленій статті зроблено спробу висвітлити історичні факти, які довгий час перебували в тіні, як маловідомий польський професор із Львівського університету Александр Завадські допоміг непомітному австрійському ченцю Менделю започаткувати основи генетики.Общепризнано, что Иоганн Грегор Мендель является основателем генетики – науки, изучающей важнейшее для понимания всех жизненных процессов явление – наследственность.Однако как генетик, находящийся под влиянием моих учителей во Львове и Гданьске, Копенгагене и Коулд Спринг Харборе (Нью- Йорк), я всегда удивлялся, как удалось Менделю быть таким независимым мыслителем при отсутствии у него наставника, способного направить его открытия и аналитический ум? Был ли кто-нибудь в жизни Менделя, кто занимал такое положение? В крайнем случае, была ли у него возможность обсуждать свои эксперименты с кем-нибудь из коллег и получать помощь от руководителя или друга при анализе полученных результатов? В представленной статье сделана попытка осветить долгое время находившиеся в тени истории факты, как малоизвестный польский профессор Львовского университета Александр Завадски помог незаметному австрийском монаху Менделю создать основы генетики

Laying the foundations for a bio-economy

Biological technologies are becoming an important part of the economy. Biotechnology already contributes at least 1% of US GDP, with revenues growing as much as 20% annually. The introduction of composable biological parts will enable an engineering discipline similar to the ones that resulted in modern aviation and information technology. As the sophistication of biological engineering increases, it will provide new goods and services at lower costs and higher efficiencies. Broad access to foundational engineering technologies is seen by some as a threat to physical and economic security. However, regulation of access will serve to suppress the innovation required to produce new vaccines and other countermeasures as well as limiting general economic growth

Dynamic assembly of primers on nucleic acid templates

A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of ∼1 μM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy

Yeast:One cell, one reference sequence, many genomes?

The genome of Saccharomyces cerevisiae – brewer’s or baker’s yeast – was the first eukaryotic genome to be sequenced in 1996. The identity of that yeast genome has been not just a product of sequencing, but also of its use after sequencing and particularly of its mobilization in scientific literature. We ask “what is the yeast genome?” as an empirical question by investigating “the yeast genome” as a discursive entity. Analyzing publications that followed sequencing points to several “yeast genomes” existing side-by-side: genomes as physical molecules, digital texts, and a historic event. Resolving this unified-yet-multiple “genome” helps make sense of contemporary developments in yeast genomics such as the synthetic yeast project, in which apparently “the same” genome occupies multiple roles and locations, and points to the utility of examining specific non-human genomes independent of the Human Genome Project

A One Pot, One Step, Precision Cloning Method with High Throughput Capability

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation

Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer

The FokI restriction endonuclease is a monomeric protein that recognizes an asymmetric sequence and cleaves both DNA strands at fixed loci downstream of the site. Its single active site is positioned initially near the recognition sequence, distant from its downstream target 13 nucleotides away. Moreover, to cut both strands, it has to recruit a second monomer to give an assembly with two active sites. Here, the individual steps in the FokI reaction pathway were examined by fluorescence resonance energy transfer (FRET). To monitor DNA binding and domain motion, a fluorescence donor was attached to the DNA, either downstream or upstream of the recognition site, and an acceptor placed on the catalytic domain of the protein. A FokI variant incapable of dimerization was also employed, to disentangle the signal due to domain motion from that due to protein association. Dimerization was monitored separately by using two samples of FokI labelled with donor and acceptor, respectively. The stopped-flow studies revealed a complete reaction pathway for FokI, both the sequence of events and the kinetics of each individual step