# Comptes Rendus
Biologies

Adrian Minty

**On learning molecular biology in François Gros' lab in the late 1970s and early
1980s**

Volume 346, Special Issue S2 (2023), p. 37-40

Online since: 21 December 2023

Issue date: 29 March 2024

**Part of Special Issue:** A tribute to François Gros, a founding father of molecular biology

**Guest editors:** Margaret Buckingham (Professeur émérite et directeur honoraire du département Biologie du développement et cellules souches à l'Institut Pasteur - Membre de l'Académie des sciences) and Moshe Yaniv (Professeur honoraire à l'Institut Pasteur - Directeur de recherche émérite au CNRS - Membre de l'Académie des sciences)

https://doi.org/10.5802/crbiol.134

<figure><img src="image_1.png" /></figure>
A tribute to François Gros, a founding father of molecular biology

# On learning molecular biology in François Gros' lab
in the late 1970s and early 1980s

Adrian Minty<sup>a</sup>

<sup>a</sup> Sanofi Recherche, Toulouse, France (retired)

E-mail: adrianjohnminty@gmail.com

**Abstract.** Although reflection is obviously crucial in molecular biology, experimentation is nonethe-
less the basis of most major advances. I was lucky to begin my research career at a particularly inter-
esting time, and privileged to have spent a number of years in François Gros' laboratory at the Institut
Pasteur. His influence, and that of his lab, were crucial in shaping my early career.

**Keywords.** mRNA, Molecular biology, Gene cloning, Gene expression.

Published online: 21 December 2023, Issue date: 29 March 2024

1. On the discovery of mRNA and my interest in it

The idea of an unstable messenger RNA taking infor-
mation from the genome to the sites of protein syn-
thesis now seems to us to be obvious. To see that this
was not the case at the time of the experiments of
François Gros and colleagues in 1961 [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 [2].
In this book, Professor Harris re-examines in detail
the critical experiments of Gros *et al*. [1] and others,
and continues to conclude that it is, in fact, riboso-
mal 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 undergradu-
ate 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 François at the Institut Pasteur.

2. On RNA populations in animal cells and gene number

Now that RNA could be isolated, our work at the Beat-
son Institute, and parallel studies by Nabeel Affara in
François' lab in Pasteur, by John Bishop's lab in Edin-
burgh, Eric Davidson's lab in LA and others, was to at-
tempt to devise experimental ways of studying RNA
populations in animal cells. We used the newly dis-
covered 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 es-
timate 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 [3]. This is in the same range as the figure
deduced twenty years later for the number of human
genes by genome sequencing [4].
**3. On non-polyadenylated mRNA**

In 1978, incited by my friend Nabeel Affara, I moved to the Institut Pasteur to work with François. I still remember one of my early exchanges with François, 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) or 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 [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 *et al*. [6]. Few other studies have been done since on this question, but those that have been performed support our conclusion [7].

**4. On the advent of gene cloning**

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 François’ laboratory was no exception (see his chapter on *Le "boum" du génie génétique* [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 crossing the rue du Dr. Roux to discuss with François 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 [9]. This work enabled us to characterise, for the first time, the expression of both skeletal muscle and cardiac actins in skeletal muscle [10].

**5. On the retro-transcription of polyadenylated mRNA and integration of DNA copies in the genome**

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 [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 [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 [13], quoted Professor Michel Goldman of the Université 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 [14]. When we combat conspiracy
theories on the potential dangers of RNA vaccines,
this should not involve ignoring any potentially con-
tradictory facts!

**6. On the nature gene regulatory sequences**

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 lab-
oratory 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 tran-
scription start site for the human cardiac actin gene,
I was to have some luck.

This was the time in 1984 when Apple was launch-
ing 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 compar-
ison programme, that the “CCArichGG” regulatory
sequence was identified at my house in Mountain
View [15]. These CCArGG DNA sequences have since
been shown to be more widespread than we orig-
inally 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 [16]. I feel extremely lucky to have been able to
participate, thanks to François’ help, in this very ex-
citing period of research.

Declaration of interests

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

Acknowledgements

It will be clear from the above, that the early years of
my research career were greatly shaped by my time
in François Gros' lab. I will always be grateful to
François 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 François' 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!

References

[1] F. Gros, H. Hiatt, W. Gilbert, C. G. Kurland, R. W. Riesbrough, J. D. Watson, "Unstable ribonucleic acid revealed by pulse labelling of Escherichia coli?", *Nature* **190** (1961), p. 581-585.

[2] H. Harris, *Nucleus and Cytoplasm*, 2nd ed., Clarendon Press, Oxford, 1970.

[3] A. J. Minty, G. D. Birnie, "Messenger RNA populations in eu-
karyotic cells—evidence from recent nucleic acid hybridiza-
tion experiments bearing on the extent and control of differ-
ential gene expression", in *Biochemistry of Cellular Regula-
tion* (M. A. Buckingham, ed.), Development and Differentia-
tion, vol. III, CRC Press, Boca Raton, FL, 1981, p. 43-82.

[4] E. S. Lander *et al.*, "Initial sequencing and analysis of the human genome", *Nature* **409** (2001), p. 860-921.

[5] A. J. Minty, F. Gros, "Coding potential of non-polyadenylated messenger RNA in mouse Friend cells", *J. Mol. Biol.* **139** (1980), p. 61-83.

[6] J. E. Darnell, H. Lodish, D. Baltimore, *Molecular Cell Biology*, Scientific American, New York, 1986.

[7] L. Yang, M. O. Duff, B. R. Graveley, G. C. Carmichael, L.-L. Chen, "Genomewide characterisation of non-polyadenylated RNAs", *Genome Biol.* **12** (2011), article no. R16.

[8] F. Gros, *Les Secrets du Gène*, Editions Odile Jacob, Paris, 1986.

[9] A. J. Minty, M. Caravatti, B. Robert, A. Cohen, P. Daubas, A. Weydert, F. Gros, M. E. Buckingham, "Mouse actin mes-senger RNAs. Construction and characterization of a recom-binant plasmid molecule containing a complementary DNA transcript of mouse alpha-actin mRNA", *J. Biol. Chem.* **256** (1981), p. 1008-1014.

[10] A. J. Minty, S. Alonso, M. Caravatti, M. E. Buckingham, "A fetal skeletal actin mRNA in the mouse and its identity with cardiac actin mRNA", *Cell* **30** (1982), p. 185-192.

[11] A. J. Minty, S. Alonso, J. L. Guenet, M. E. Buckingham, "Num-
ber and organization of actin-related sequences in the mouse
genome", *J. Mol. Biol.* **167** (1983), p. 77-101.

[12] Z. Zhang, P. M. Harrison, M. Gerstein, "Millions of years of evolution preserved: a comprehensive catalog of processed pseudogenes in the human genome", *Genome Res.* **13** (2003), p. 2541-2558.

[13] A. Whiting, "Five things you need to know about: mRNA vaccine safety", in *Horizon (The EU Research & Innovation Magazine)*, European Commission, Directorate-General for Research and Innovation, Brussels, 2020.

[14] T. Domazet-Loso, "mRNA vaccines: Why is the biology of retroposition ignored?", *Genes* **13** (2022), p. 719-754.
[15] A. Minty, L. Kedes, "Upstream regions of the human cardiac actin gene that modulate its transcription in muscle cells: presence of an evolutionarily conserved repeated motif", *Mol. Cell Biol.* **6** (1986), p. 2125-2136.

[16] S. Kappel, F. Rumpler, G. Theisssen, "Cracking the floral quar-

tet code: How do multimers of MIKC<sup>C</sup> type MADS-Domain transcription factors recognize their target genes?", *Int. J. Mol. Sci.* **24** (2023), article no. 8253.