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\DOI{10.5802/crbiol.134}
\datereceived{2023-09-29}
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\dateposted{2023-12-21}
\datepublished{2024-03-29}
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%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{A tribute to Fran\c{c}ois Gros, a founding father of molecular biology}
%%\def\cdr@specialtitle@french{Un hommage \`a Fran\c{c}ois Gros, un des p\`eres de la biologie mol\'eculaire}
%\makeatother

\title{On learning molecular biology in Fran\c{c}ois Gros' lab in the
late 1970s and early 1980s}

\alttitle{Apprendre la biologie mol\'eculaire dans le laboratoire de
Fran\c{c}ois Gros \`a la fin des ann\'ees 1970 et au d\'ebut des
ann\'ees 1980}

\author{\firstname{Adrian} \lastname{Minty}}
\address{Sanofi Recherche, Toulouse, France (retired)}
\email{adrianjohnminty@gmail.com}

\keywords{\kwd{mRNA}\kwd{Molecular biology}\kwd{Gene cloning}\kwd{Gene
expression}}

\altkeywords{\kwd{ARN messager}\kwd{Biologie
mol\'{e}culaire}\kwd{Clonage de g\`{e}nes}\kwd{Expression de
g\`{e}nes}}

\begin{abstract} 
Although reflection is obviously crucial in molecular biology,
experimentation is nonetheless the basis of most major advances. I was
lucky to begin my research career at a particularly interesting time,
and privileged to have spent a number of years in Francois Gros'
laboratory at the Institut Pasteur. His influence, and that of his
lab, were crucial in shaping my early career.
\end{abstract}

\begin{altabstract}
Si la r\'{e}flexion est \'{e}videmment cruciale en biologie
mol\'{e}culaire, l'exp\'{e}rimentation est n\'{e}anmoins \`{a} la base
de la plupart des grandes avanc\'{e}es. J'ai eu la chance de commencer
ma carri\`{e}re de chercheur \`{a} une \'{e}poque particuli\`{e}rement
int\'{e}ressante, et le privil\`{e}ge de passer plusieurs ann\'{e}es
dans le laboratoire de Fran\c{c}ois Gros \`{a} l'Institut Pasteur. Son
influence et celle de son laboratoire ont \'{e}t\'{e} d\'{e}terminantes
pour le d\'{e}but de ma carri\`{e}re. 
\end{altabstract}

\maketitle

{\vspace*{25pt}}

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\section{On the discovery of mRNA and my interest in it}\label{sec1}

The idea of an unstable messenger RNA taking information from the
genome to the sites of protein synthesis now seems to us to be obvious.
To see that this was not the case at the time of the experiments of
Fran\c{c}ois Gros and colleagues in 1961~\cite{1}, readers are
encouraged to have a look at the second edition of the book Nucleus and
Cytoplasm, published by Professor Henry Harris of the University of Oxford
in 1970~\cite{2}. In this book, Professor Harris re-examines in detail the
critical experiments of Gros \etal~\cite{1} and others, and continues
to conclude that it is, in fact, ribosomal RNA which is the messenger!

When I was completing my biochemistry degree at Cambridge in 1974, the
subject of mRNA was still a hot one, and I was lucky to do an
undergraduate project on RNA translation in reticulocyte lysates with
Tim Hunt and Richard Jackson. This persuaded me to continue in the
field of mRNA for my PhD, at the Beatson Institute in Glasgow, and
subsequently in my postdoc with Fran\c{c}ois at the Institut Pasteur.
\vspace*{21pt}

\section{On RNA populations in animal cells and gene number}\label{sec2}

Now that RNA could be isolated, our work at the Beatson Institute, and
parallel studies by Nabeel Affara in Fran\c{c}ois' lab in Pasteur, by
John Bishop's lab in Edinburgh, Eric Davidson's lab in LA and others,
was to attempt to devise experimental ways of studying RNA populations
in animal cells. We used the newly discovered reverse transcriptase
enzyme to make copies of all polyadenylated mRNAs. In highly
miniaturised experimental ``containers'' with microlitre volumes, we
mixed the RNA and radioactively labelled DNA copies and then studied
the formation of RNA--DNA duplexes over times up to one week.

The results of these experiments allowed us to estimate the number of
genes expressed in an animal cell, and the number of these genes in
common with a second cell type. Given all the uncertainties in the
calculations, the results were surprisingly consistent from one study
to another, with most animal cells and tissues expressing between
10,000 and 20,000 genes~\cite{3}. This is in the same range as the
\mbox{figure} \mbox{deduced} twenty years later for the number of human
genes by genome sequencing~\cite{4}.

\section{On non-polyadenylated mRNA}\label{sec3}

In 1978, incited by my friend Nabeel Affara, I moved to the 
Institut Pasteur to work with Fran\c{c}ois. I still remember one of my early
exchanges with Fran\c{c}ois, who was a very good letter writer, which
took place while I was still in Glasgow. He expressed the hope that an
``excellent articulation'' would be established between us. I like to
think that this was the case!

At the Institut Pasteur, I continued the study of mRNA. In all the
previously mentioned studies on mRNA, we had experimentally defined
mRNA as being polyadenylated, since either oligo(dT)-cellulose or
poly(U)-Sepharose chromatography were used to isolate it. However, a
significant fraction of mRNA (defined by its translation in a
reticulocyte lysate) did not bind to oligo(dT) ou poly(U). We therefore
attempted to see if this fraction encoded different proteins, and thus
represented a different type of messenger RNA.

Analysing translation products of highly purified poly(A)$^{+}$ and
poly(A)$^{-}$ preparations on two-dimensional gel electrophoresis, we
showed that the two populations encoded similar proteins, but in very
different abundancies~\cite{5}. One protein class which was almost
entirely poly(A)$^{-}$ was that encoding the histones. Our studies
showed that, apart from the histones, poly(A)-mRNA does not encode
another, particular, subset of mRNAs. This work, and the critical
2D-gel figure, were cited in the Molecular Cell Biology book by Darnell
\etal~\cite{6}. Few other studies have been done since on this
question, but those that have been performed support our
conclusion~\cite{7}.

\section{On the advent of gene cloning}\label{sec4}

The previous studies on mRNA populations represented the last in the
``pre-cloning era''. The advent of recombinant DNA technology was to
revolutionise our experimental approaches in molecular biology, and
Fran\c{c}ois' laboratory was no exception (see his chapter on
\textit{Le ``boum'' du g\'{e}nie g\'{e}n\'{e}tique}~\cite{8}). A gene
cloning project was set up in Margaret Buckingham's group, and I took
the responsibility for cloning the actin genes of the mouse. I~still
remember \mbox{crossing} the rue du Dr. Roux to discuss with Fran\c{c}ois
Rougeon, one of the world specialists in gene cloning, who warned me
that this might be complicated because the actin mRNAs were less
abundant than the globin mRNA and the other mRNAs that had been cloned
up to then! Consequently, we subjected the RNA to sucrose-gradient
separation to enrich for the actin mRNA(s) before cloning!

Gene cloning was something of a challenge for someone who had not
previously done experimental work with bacterial plasmids, nor indeed
with bacteria! Nonetheless, the abundance of actin mRNAs (particularly
in the size-purified RNA!) meant that a hundred or so recombinant
bacteria were largely sufficient to isolate several different actin
cDNAs, including two different muscle actin cDNAs~\cite{9}. This work
enabled us to characterise, for the first time, the expression of both
skeletal muscle and cardiac actins in skeletal muscle~\cite{10}.

\section{On the retro-transcription of polyadenylated mRNA and
integration of DNA copies in the genome}\label{sec5}

One of the first subjects to be addressed with the newly cloned actin
cDNAs, was the number of actin genes in mammalian genomes. We found
that in mouse DNA, in addition to the six actin genes expected from
protein sequencing work, there was also a family of weakly-related
sequences, some of which had recently been amplified in the
genome~\cite{11}. Although these had not been sequenced, the length of
these actin-like genes in genomic DNA led us to predict that they could
represent intron-less pseudogenes, generated by reverse transcription
of actin mRNAs and insertion in the genome. This process has since been
confirmed, with the sequencing of over 8000 retropseudogenes in the
human genome~\cite{12}.

It is somewhat surprising that studies on mRNA-copy reintegration into
the genome were largely ignored during the recent debate on the
possibility of Covid-19 RNA vaccine reintegration in the genome. Alex
Whiting, writing in 2020~\cite{13}, quoted Professor Michel Goldman of the
Universit\'{e} Libre de Bruxelles as saying ``mRNA vaccines do not
alter your DNA. A concern that some have had about mRNA vaccines is
that they could change people's DNA. But that idea is `completely
false' and has `no scientific basis'.''

It would seem, in fact, that it is stating the impossibility of
re-integration into the genome which is completely false~\cite{14}.
When we combat conspiracy \mbox{theories} on the potential dangers of
RNA vaccines, this should not involve ignoring any potentially
contradictory facts!

\section{On the nature gene regulatory sequences}\label{sec6}

The ``grail'' of molecular biologists in the 1980s was the
identification of the sequences regulating gene expression in mammalian
cells. In pursuit of this, I was to continue the work on actin genes in
the laboratory of Larry Kedes at Stanford University. Apart from the
9000~km that separated Paris from Palo Alto, the main
difference between the two labs was that the Kedes lab worked on human
actin genes rather than mouse actin genes. After some months of rather
painstaking experimental work identifying the transcription start site
for the human cardiac actin gene, I was to have some luck. 

This was the time in 1984 when Apple was launching the first Apple
Macintosh, and a special offer was made to workers at Stanford. It was
thus on my own Macintosh, using a very rudimentary DNA comparison
programme, that the ``CCArichGG'' regulatory sequence was identified at
my house in Mountain View~\cite{15}. These CCArGG DNA sequences have
since been shown to be more widespread than we originally anticipated,
and to have more diverse roles. Indeed, the molecular mechanisms of the
binding of transcription factors to these sequences are still in the
process of being elucidated thirty-seven years later~\cite{16}. I feel
extremely lucky to have been able to participate, thanks to
Fran\c{c}ois' help, in this very exciting period of research.

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Acknowledgements}

It will be clear from the above, that the early years of my research
career were greatly shaped by my time in Fran\c{c}ois Gros' lab. I will
always be grateful to Fran\c{c}ois for allowing me to come and work in
his lab, for allowing me to choose my research subjects and for
offering me so often some of his precious time and advice. Among the
other people who helped me a lot during my time at Pasteur, I would
also like to thank Nabeel Affara, who persuaded me to come to Paris,
and Margaret Buckingham with whom I was to work on the gene cloning
project. Last, but very decidedly not least, it was also working in
Fran\c{c}ois' lab that I was to meet my wife Catherine, and it was
there too that I acquired the nationality of a country that has now
been mine for the last forty-five years!

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