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Where it all happened - the locations where people have worked with lampbrush chromosomes worldwide since 1882


 


Recent and current work in the Gall laboratory (2014) in Baltimore MD

Our lab continues to study both the structure of LBCs and the RNA transcribed from them, using the favorable chromosomes of the newt Notophthalmus viridescens and the axolotl Ambystoma mexicanum for most of our structural studies, and the RNA from GVs of the frogs Xenopus tropicalis and X. laevis for our molecular analyses.

Over the past few years we have used super-resolution microscopy to examine individual transcription units of Notophthalmus in greater detail than is possible by confocal microscopy. Depending on the type of microscope used, we obtain resolutions in the range of 50-100 nm, compared to 200 nm for confocal. Although this resolution does not match what is possible by electron microscopy, the great advantage of the newer microscopes is that they are based on fluorescence. Thus, the full range of fluorescent tags and immunofluorescent antibodies is available.

Using antibodies against RNA polymerase II we see increased detail in the axes of individual LBC loops. Specifically, pol II is not uniformly distributed along the axis. Instead, there are short regions with polymerase staining interspersed with regions that lack polymerase. It is not clear, of course, how much of this pattern might be preparative artifact, but it is possible that there are regions where the polymerase moves rapidly along the axis and regions where it pauses.

We used the brilliant staining of an antibody against CELF1 protein to look at details of the nascent transcripts (see the 2007 Chromosome Research paper by Garry Morgan for why this protein is valuable for analyzing LBC loops). Super-resolution reveals that the coarsely granular pattern of CELF1 staining on certain loops is due to what appears to be rather uniform particles with a diameter no greater than about 100 nm. Again, the issue of artifacts must be considered, both from sample preparation and possibly from the algorithms used to generate the images. Our first publication using super-resolution microscopy appeared in Chromosome Research in 2012 (Kaufmann, Cremer, and Gall).

To complement our structural analysis of LBC loops, we embarked several years ago on a program to analyze the transcriptome of the oocyte by deep sequencing. We chose Xenopus tropicalis for this purpose, because its genome has been sequenced and annotated in greater detail than that of any other amphibian. A unique advantage of the Xenopus oocyte is that pure samples of cytoplasmic and nuclear RNA can be obtained by manual dissection, something that is almost impossible with any other cell type. We found that cytoplasmic RNA, as expected, contains spliced transcripts derived from thousands of genetic loci. Nuclear RNA, on the other hand, was a complete surprise. We expected to see nascent transcripts or partially processed pre-mRNA molecules (in addition to ribosomal RNA precursors and small RNAs of various sorts). Instead we found an abundance of stable RNAs derived from the introns of the same genes represented in the cytoplasm. We refer to this population as stable intronic sequence (sis) RNA (Gardner et al. 2012). Surprisingly, we find a population of sisRNA molecules in the cytoplasm of the oocyte, all of which are circular (lariats without tails) (Talhouarne and Gall 2014). We find that sisRNA is transmitted intact to the early embryo and persists through early embryogenesis until at least the blastula stage. So far we do not know the function(s) of sisRNA.

In retrospect, we should not have been surprised that we did not detect nascent transcripts in our GV RNA samples. Although transcription per gene is very high at the LBC stage, the GV contains only four copies of each gene (two in each homologue). Thus the total amount of RNA present as nascent transcripts in a single GV is vanishingly small, and our RNA samples are derived from only a few hundred hand-isolated GVs. By careful bioinformatic analysis, Zehra Nizami in our lab has begun to detect the minor fraction of nascent transcripts in our GV RNA samples. Eventually we hope to characterize these transcripts and to correlate our sequence data with what can be seen on the LBCs by in situ hybridization.


 

A remarkable career in science—Joseph G. Gall

Endow SA, Nizami ZF and Gerbi SA (2013) Chromosome Research 21: 339-343 (PDF)

A festive group of ~150 current and former students, postdoctoral and other associates, and colleagues gathered during the weekend of April 12–14, 2013 to celebrate Joe Gall's 85th birthday. The gathering, hosted by the Carnegie Institution for Science, Department of Embryology (Allan Spradling, Director) and organized by a group of Joe's current and former students (Zehra Nizami, Alison Singer, Ji-Long Liu, Virginia Zakian, Susan Gerbi), was held in Baltimore, MD. Dinners and symposia extending over 3 days celebrated Joe's scientific findings over the years, together with those of his former students, postdoctoral fellows, and other associates.


Oscar Miller (1925 – 2012) – the technological genius of chromosome science


Oscar and his wife Mary-Rose 1973

Oscar Miller died on January 28, 2012 at the University of Virginia Hospital.
He was born on April 12, 1925 in Gastonia, North Carolina. He served in the US Navy from 1943 through 1946. After earning a bachelor's and master's degrees in agronomy from North Carolina State University, he was a farmer for six years and then enrolled in the University of Minnesota where he was awarded a PhD in plant genetics. He joined the research staff at Oak Ridge National Laboratory in 1961 and began the work that earned him worldwide renown as a molecular biologist.
Oscar was elected to the National Academy of Sciences in 1978. He was named a Fellow of the American Association for the Advancement of Science in 1980. He was Chairman of the Department of Biology and held the William R. Kenan and the Lewis and Clark Professorship at the University of Virginia. He was visiting professor of biology at the Center of Investigation and Advanced Studies in Mexico City, the California Institute of Technology, the Max-Planck-Institut für Zellbiologie in Heidelberg, Germany and the University of California at Irvine. He was the Senior Fulbright Scholar at the Division of Molecular Biology, CSIRO at New South Wales, Australia. Among his many honours from around the world, he received the Life Achievement Award in Science from the Commonwealth of Virginia in 1997.
Prior to Oscar Miller's time, the existence of genes and of the double helical structure of DNA were accepted in the scientific community as sound hypotheses but what genes did and how they did it in terms of transcription was beyond the scope of actual observation. Then Oscar developed a technique – now known as "Miller spreading"- that enabled scientists, through electron microscopy, to visualize individual genes caught in the very act of making their RNA transcripts and this, of course, opened the door to many important advances in chromosome and nuclear science.
I first met Oscar in 1969, shortly after he had developed and published his spreading technique. I remember being utterly humbled and overawed by the extraordinary skill, ingenuity, inventiveness and courage behind the work. His method is described in his 1969 Science paper (with Barbara Beatty) as follows:
The specimen was prepared by placing the contents of an oocyte nucleus in deionised water, thus causing dispersal of the nucleolar components; the unwound cores were centrifuged through a neutral solution of 0.1 molar sucrose with 10% formalin onto a carbon covered grid; the grid was rinsed in 0.4% Kodak Photo-Flo solution before drying; the preparation was then stained for 1 minute with 1% phosphotungstic acid in 50% ethanol at pH 2.5 (unadjusted), rinsed in 95% and then 100% ethanol, and dried with isopentane. (Miller, O.L. and Beatty B.R. 1969. Visualization of nucleolar genes. Science N.Y. 164, 955 – 957)


An electron micrograph of a single nucleolar gene transcription unit prepared by the Miller spreading technique. c: non-transcribing chromatin of the spacer region. p: the position of the first polymerase molecules at the start of the transcription unit. t: closely packed RNA transcripts extending sideways from the DNA axis. e: the end of the transcription unit.

Modern young chromosome scientists can have no concept of the reality of the process outlined in this brief description. In culinary terms, it was like making the world's most delicious cake "from absolute scratch". First, isolate the oocyte nucleus - not the easiest thing to do. Isolate it in deionised water – well, what you don't know is that it immediately disappears, so what follows is done on faith. Centrifuge the contents through a sucrose/formalin mix – lots of ingenuity and testing to find the right concentrations. Centrifuge the contents onto an electron microscope grid. Well, the grid is 3mm in diameter and extremely delicate and the whole business depends on scrupulous attention to detail and cleanliness – and again, lots of trial and error. Carbon covered grid – you could spend days learning to make really good carbon coated grids! Besides, centrifuge - with what? You couldn't buy a centrifuge for spinning DNA onto grids – you had to make one. And when you had done that you had find out how fast to spin and how long to spin and you had to find out the hard way that if you started the spin too fast or too slow, everything landed up in a heap at one edge of the grid. We tried to extend Oscar's work to look at transcription on the loops of the lampbrush chromosomes in amphibian oocytes and we discovered just how much skill and patience is needed to produce meaningful results – and we had the benefit of tapping into Oscar's experience. I remember well my first reaction when I saw the pictures of his transcribing genes and saw how he had done it: this man was truly a technological genius. Since its original publication in 1969 the Miller and Beatty paper has been cited nearly 1000 times. In terms of "impact" in the field of chromosome and nuclear science it is almost unrivalled.
The Oscar I remember was a great character - in that smooth, quiet, Southern and utterly charming way, all on top of a supreme competence as a scientist and teacher. Those of us who knew him will feel a sense of privilege and pleasure. Young chromosomologists the world over should take time to look at some of his work and perhaps aspire to be just a little bit like him. Science "from scratch" is fun and Oscar was one of the best at doing this kind of thing.

Herbert Macgregor



 

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