Hildegard Lamfrom helped show where proteins come from! The other day I told you about Katalin Karikó, who figured out how to make modify synthetic mRNA so that it could be effectively used to deliver protein instructions to cells, paving the way for (among other things) today’s mRNA-based vaccines. Today I want to tell you about another female scientist who was crucial to today’s successes –  Hildegard Lamfrom, who was one of the first people to provide evidence that messenger RNA copies of protein recipes serve as an intermediary between genes and proteins. And one of the first to show that the protein-making complexes called ribosomes kinda cluster together into “polyribosomes” where there are lots of ribosomes simultaneously working on the same mRNA, traveling along it and piecing together the indicated amino acids (protein letters) into growing peptide chains in a process called translation. Lamfrom was able to do much of this work by helping develop one of the first “in vitro translation” systems (ways to make proteins outside of cells) and used it to make lots of discoveries about the process of protein-making (translation).

video added 2/27/22

This is part of a post I made last September, when I created a Wikipedia page for her which I hope you’ll check out and improve (because anyone can edit Wikipedia!). Here’s the blog form of that post, which includes information about Wikipedia editing:  http://bit.ly/hildegardlamfromwikipedia

Hildegard was born in Augsburg, Germany in 1922. Her family was Jewish and fled Nazi persecution when Hildegard was 15, coming to the US and establishing themselves in Portland, Oregon. Money was tight, so when Hildegard went to college (at Reed) to study biology she financed her education by working as a welder in the shipyards. She then got a master’s in biology and got accepted into what’s now Case Western Reserve University’s medical school. But she was drawn to research, so decided to go for the PhD instead. And she chose quite the icon of an advisor – Harry Goldblatt, a renowned renal pathologist (studier of kidney disease). She got her PhD in 1949 for work done characterizing the renin-antirenin system, which helps regulate blood pressure. And then she followed Goldblatt to California and kept working with him for 5 years at Cedars of Lebanon Hospital in Los Angeles. From there, she did research all around the world – Denmark, California, England, France, Oregon, California, Boston. And she even set up a research laboratory called “Biocenter’  in India with her close friend and colleague Anand Sarabhai.

This world-traveler was loved by those around the world whom she mentored, including Brian Druker of Gleevec fame. Yet not enough people know her name! So let’s change that – because she did some remarkable work. I will tell you just a brief bit, but to get you to truly appreciate it, I need to set up the story a little.

Scientists these days know a lot about how proteins get made, a process called translation. The “recipes” for making proteins are written in stretches of DNA called “genes.” Then, when a cell wants to make a protein, it first makes a messenger RNA (mRNA) copy of that gene in a process called transcription. That mRNA gets passed off to protein-making complexes called ribosomes which travel along the mRNA and use its instructions to link amino acids (protein “letters”/“building blocks”) together through peptide bonds to form long polypeptide chains that fold up into beautiful, functional proteins. The ribosome knows what to link because as it travels along the mRNA, it pauses briefly on each 3-RNA letter combo (called a codon) and a molecule called a transfer RNA with the complementary 3-letter combo (called the anticodon) brings it the corresponding letter. So, for example, when the ribosome gets to “CAG,” a transfer RNA will bring it a glutamine.  And when it gets to “ACG” a different tRNA will bring it a different amino acid, threonine. more here: https://bit.ly/translationtimestwo 

But normally all of this is happening inside of cells, which makes it really hard to figure out what’s going on. So scientists who were trying to work out all that stuff wanted to see if they could recreate the process outside of cells, “in vitro.” One problem was they didn’t know what they needed! So they tried breaking open (lysing) various cell types and taking the liquid part (the lysate) and seeing if it would make protein (they often added radioactive versions of amino acids so they could tell if they were). Different groups of people were working on this research, and using different cell types, etc. So I’m definitely not implying Hildegard should get all the credit here, but she, working with Richard Schweet at Caltech, and later with Paul Knopf at MRC, helped develop one of the first in vitro translation systems, using rabbit reticulocyte lysate. more on in vitro translation systems: https://bit.ly/cellfreeexpression

Reticulocytes are a form of immature red blood cells – and they made (and still make) a great system for protein synthesis for a couple reasons. The main function of red blood cells is to carry oxygen throughout your body on the backs of hemoglobin protein molecules. So mature red blood cells need to have a lot of hemoglobin and not much else. Therefore, during the maturation process, they lose a lot of cell components that just “get in the way” – they even loose their nucleus (the membrane-bound compartment of the cell where DNA is stored). But they keep hemoglobin mRNAs and all the protein-making equipment. So if you break them open (lyse them) and get rid of the membrane-y stuff, you have a lysate that’s capable of protein synthesis.

And, if you just add some amino acids, the protein it’s gonna synthesize is hemoglobin, which makes up >90% of the protein that gets made in these cells, so it’s the predominant mRNA that’s present. Now, if you want them to make something other than hemoglobin, you’re in trouble (which is why Hugh Pelham and Richard Jackson later established a still-widely-use protocol where you add micrococcal nuclease, which is an RNA chewer which will degrade all the hemoglobin mRNA (and any other mRNA transcripts) in the lysate – and then you add the mRNA for the protein you want). https://pubmed.ncbi.nlm.nih.gov/823012/

But at this point in time, scientists still didn’t even know if mRNA even existed! So what did they know? For one thing, they knew that proteins were made by ribosomes (which they often refer to as microsomes). Hildegard was one of the first to show that, what some people thought were just “aggregates” of clumped-up ribosomes were actually the site of protein synthesis. We now know that these “polyribosomes” (aka polysomes) consist of multiple ribosomes working on the same mRNA at the same time.

Hildegard, working with Paul Knopf, was able to separate the individual ribosomes (monosomes) from the groups of ribosomes (polysomes) based on their size (using a density gradient centrifugation and seeing how far they sink in a bed of sucrose). When they added radioactive amino acids, they saw radioactivity in the polysome fraction and then it would shift to the liquid fraction as the (now-radioactive) hemoglobin was released from those polysomes. And when they added radioactive ribosomes, they saw the radioactivity shift from the monosomes from the polysomes, demonstrating that new monosomes can join in. https://doi.org/10.1016/S0022-2836(64)80227-8

Speaking of separating things, in Hildegard’s early work with the rabbit reticulocyte lysate system, she was separating various fractions of the lysate and then mixing them together. So, for instance, she separated the ribosomes in one fraction and some other stuff in a different fraction. When she mixed the right components she could get the ribosomes to finish making what they were already working on, but they were having trouble starting new proteins. Turns out that the mRNA was getting fragmented during the prep, and, when she switched to using the whole lysate, she got better results and was able to show (by labeling the end) that brand-new hemoglobin proteins could get made.

But why was hemoglobin getting made? How did the ribosomes know to make it? This is before mRNA was a proven thing – Jacob et. al had theorized its existence, but there wasn’t direct experimental evidence that the specific proteins which a ribosome would make was dictated by mRNA.

Hildegarde was one of the scientists who wanted to find out what determines which protein a ribosome makes. In the reticulocyte lysate, it could be easy to think that the ribosomes were making hemoglobin because that was all they could make – maybe it was something inherent in those ribosomes (e.g. maybe your cells make one ribosome specialized in making hemoglobin and another ribosome specialized in making keratin, and your blood cells make a lot of the hemoglobin-makers whereas your skin makes a bunch of the keratin-makers). If that were the case, no matter where you put it, a ribosome would only make the exact same protein as it was “born to make.” BUT, if the ribosome’s fate was not pre-determined, you should be able to get it to make something different. And Hildegarde used a clever experiment to show that this was indeed the case.

She purified ribosomes from a sheep and ribosomes from a rabbit. Then she mixed the sheep ribosomes with ribosome-less rabbit reticulocyte lysate. And she did the opposite, mixing rabbit ribosomes with sheep reticulocyte lysate. And then she looked to see what got made. And she saw a mix of rabbit and sheep hemoglobin in each case. She interpreted this as being because some of the ribosomes had some host mRNA attached so they could make that (e.g. sheep ribosomes on sheep hemoglobin mRNA would make sheep hemoglobin) but they could also make the rabbit hemoglobin using the rabbit hemoglobin mRNAs in the lysate. So, it wasn’t the ribosome in charge, there was something outside the ribosome directing the show! And this was some of the first direct experimental evidence for mRNA. https://doi.org/10.1016/S0022-2836(61)80064-8

This is some of the most exciting work she did (in my opinion) but it was far from the only work she did. Unfortunately, her life was cut short by a brain tumor, and she died in 1984 at the age of 62. Her sister Gertrude (Gert) Boyle served as the president of Columbia Sportswear and became a bit of an entrepreneurial legend. But to Gert, one of the most important things was that her sister Hildegard was remembered for the biochemical legend Hildegard was. So she donated a LOT of money to Oregon Health and Sciences University (OHSU) for cancer research and they named fellowships and buildings after her.

Key publications:

LAMFROM H, KNOPF PM. INITIATION OF HAEMOGLOBIN SYNTHESIS IN CELL-FREE SYSTEMS. J Mol Biol. 1964 Aug;9:558-75. doi: 10.1016/s0022-2836(64)80227-8. PMID: 14202286. https://doi.org/10.1016/S0022-2836(64)80227-8

LAMFROM H. Factors determining the specificity of hemoglobin synthesized in a cell-free system. J Mol Biol. 1961 Jun;3:241-52. doi: 10.1016/s0022-2836(61)80064-8. PMID: 13758530. https://doi.org/10.1016/S0022-2836(61)80064-8

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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