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One of the major results of the Human Genome Project (HGP) was that humans have far fewer separate genes than previously thought. From a 2004 article about the HGP:

Francis S. Collins, director of the National Human Genome Research Institute (NHGRI), said, "Only a decade ago, most scientists thought humans had about 100,000 genes. When we analyzed the working draft of the human genome sequence three years ago, we estimated there were about 30,000 to 35,000 genes, which surprised many. This new analysis reduces that number even further [to 20,000-25,000] and provides us with the clearest picture yet of our genome."

What was the old estimate of 100,000 based on? I assume that in 1994 no one had sequenced the entire proteome...

As Remi.b points out, the 100,000 gene estimate may be based on the one gene–one enzyme hypothesis. If that's true, did people before the human genome project think that there were 100,000 distinct enzyme activities? If so, what experiments/data were used to establish that number?

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  • $\begingroup$ Importance and amount of non-coding DNA was underestimated. $\endgroup$ – Mithoron Oct 13 '18 at 17:12
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There's actually no need to speculate on the answer to this question since scientists have published their estimates and methodology, as is their way. The following paper is a good review:

Fields C, Adams MD, White O, Venter JC. 1994. How many genes in the human genome? Nature Genetics 7:345-346.

Below are some truncated excerpts from the paper but, if possible, I recommend reading the whole thing and the references therein.

In genomic sequencing pilot projects... we found... an average of about one gene in 23.4 kb... Extrapolated for the whole genome, we would predict about 129,000 genes; however, the regions that we sequenced were chosen for high-GC content and hence gene richness. At most, half of the genome, in the GC rich... bands, is likely to have high gene density; if the rest has half of the density we observed, the human genome might contain 97,000 genes. But the gene-poor fraction of the genome probably has much less than half the density of the gene-rich fraction... If we assume that the genome comprises a gene-rich half with [23.4 kb per gene] and a gene-poor half with a tenth that density, we obtain an estimate of about 71,000 genes...

Making estimations based on average gene size has been discussed in another answer. The varying estimates result from the different assumptions made: there were a lot of unknowns at this time. You can read about the correlation between GC-content and gene density in this answer.

Estimates giving much lower gene content are, however, easy to come by... Wagner and colleagues note that only about 12% of a typical mammalian genome... is transcribed. Using an average gene size of 18 kb obtained from a list of characterized genes... they estimate a total of 20,000 genes. By assuming that 2,500 housekeeping genes (from estimates of Escherichia coli) constitute 18% of the total number of genes the same authors obtained an even smaller figure of some 14,000 human genes.

I don't have access to this reference so it's hard too delve to deeply into their methodology, but the number of genes expressed really depends on the cell-type. Thymic medullary cells, for example, express 85% of the coding genome. RNAseq has also suggested that over 90% of the genome is transcribed, though this controversial. All that said, their first estimate was rather spot-on.

Measurement of RNA reassociation kinetics suggest that approximately 10,000 distinct genes are expressed in a typical mammalian cell, from which Lewin estimates a total gene number of 20,000 to 40,000.

This is C0t analysis with RNA instead of DNA (called R0t). You can read more about this here. This estimate, too, has proved reasonably accurate.

Using restriction analysis with the methylation-sensitive enzyme HpaII, Antequerra and Bird estimated that the human genome contains 45,000 CpG islands. They also report that about 56% of sequenced genes contain CpG islands, and hence estimate a total gene number of about 80,000... This number, however, may be an overestimate, as even "complete" gene sequences rarely include extensive 5' or 3' flanking sequence and hence may muss associated CpG islands.

We now know that there about 30,000 CpG islands in the genome, of which about 9,000 are intragenic and that 72% of genes have CpG islands. This would revise their estimate to 30,000 genes.

We have used a collection of 3,483 nonredundant coding sequences as an effective genome against which to compare a collection of human ESTs... If [this] set of complete cDNA... is representative of human genes in general, the fraction of known cDNAs matched by randomly-selected ESTs should equal the fraction of novel sequences matched by randomly-selected ESTs. Our human EST sequencing project has so far identified ESTs matching 1,877 of the 3,483 unique coding regions (54%). We can, therefore, estimate that the novel ESTs that we have sequenced represent about 54% of previously-unknown human genes... To estimate how many genes these novel ESTs identify, we... [clustered] the ESTs. This step reduced 65,297 ESTs to 40,077 clusters... indicating that the novel EST set was 40% redundant. We can then calculate an expected number of human genes as: 40,077 / 0.54 + 3,483 = 77,700 genes. This calculation is an over-estimate, since the clustering procedure cannot identify ESTs from the same transcript unless they overlap. If the true average redundancy is 50%, we predict about 64,000 genes; if the true average redundancy is 60%, we predict 52,000 genes.

Although they accounted for alternative splicing, assumptions were made about how representative their collection of coding sequences was. It seems that one of the problems at this time was that many of the overestimates made, using different methods, more or less agree with each other. Unfortunately, many of the assumptions did not stand up.

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Human genome is 3.2Gbp (giga=billions of basepairs). If you assume there are 100k genes, this yields around 32kbp (kilo=thousands base pairs) per gene.

Before human genome project, let's say before 1990, people were isolating a lot of genes from human-derived tissues. You can use google scholar to find relevant papers. From quick search, you can see that range is pretty large:

  • "186,000 base-pair (bp) human factor VIII gene"
  • "human TF gene spans 12.4 kbp"

So you can see how one might guess "average" size of the gene to be somewhere around 30kbp, if you haven't yet found too many genes. Whereas after genome sequencing, we know that genes are 100-10000 bp long. I think the issue was that there was no enough statistics yet to judge appropriately "average gene size". And as it turns out, the distribution is very funky. You need to isolate a lot of genes to reconstruct that distribution.

By the 1990 phenomenon of the alternative splicing was already known. What perhaps wasn't quite obvious, is how many genes there are, what their real sizes, and how many genes are overlapping

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  • $\begingroup$ I think it was rather thinking that more of DNA is coding than in reality, but that's another possibility. $\endgroup$ – Mithoron Oct 13 '18 at 17:15
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    $\begingroup$ From Pertea et Salzberg, Genome Biol, 2010: An estimate of 100,000 genes appeared in the 1990 joint National Institutes of Health (NIH)/Department of Energy (DOE) report on the Human Genome Project; this was apparently based on a very rough (and incorrect) calculation that typical human genes are 30,000 bases long, and that genes cover the entire 3-gigabase genome. $\endgroup$ – tsttst Oct 16 '18 at 1:53
  • $\begingroup$ @tsttst thanks! i think it also interesting that today we can easily peek into the past literature (thanks google scholar :) $\endgroup$ – Oct18 is day of silence on SE Oct 16 '18 at 4:46
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I don't know much about the evolution of thoughts on the subject but I would suppose that the estimate of 100,000 genes is probably caused by the one gene - one enzyme/protein ideas

The one gene–one enzyme hypothesis is the idea that genes act through the production of enzymes, with each gene responsible for producing a single enzyme that in turn affects a single step in a metabolic pathway.

The idea that genes are affecting cell functions via the protein that they code for is not so outdated. However, the idea that a gene codes for a single unique protein is a little outdated.

In reality a single gene can code for several different proteins via a mechanism called alternative splicing.

Alternative splicing, or differential splicing, is a regulated process during gene expression that results in a single gene coding for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. Consequently, the proteins translated from alternatively spliced mRNAs will contain differences in their amino acid sequence and, often, in their biological functions [..]. Notably, alternative splicing allows the human genome to direct the synthesis of many more proteins than would be expected from its 20,000 protein-coding genes.

The original estimate of the number of genes was hence probably alined with the observed number of proteins by assuming that there is a one-to-one function from gene to protein (as by the one gene - one protein hypothesis).

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  • $\begingroup$ Makes sense. What I want to know though is where that particular number came from. In quantitative terms, what experiments or line of reasoning lead to 100,000? It's (basically) within an order of magnitude of the correct answer, so I'm assuming (maybe incorrectly) that the estimate was originally based on some solid numbers from somewhere. Like, was there a reason to think there were 100,000 distinct enzyme activities? $\endgroup$ – tel Oct 13 '18 at 2:11
  • $\begingroup$ The original estimate was probably based upon the number of proteins. I'll clarify that in my answer $\endgroup$ – Remi.b Oct 13 '18 at 2:48
  • $\begingroup$ Thanks for the update. There's still a missing piece, though. How did they establish 100,000 as the likely count of distinct proteins? Was there an experiment that existed in the pre-omics era that was able to resolve the presence of thousands (or 10's of thousands) of proteins in a single sample? Or was it merely an informed guess based on decades of various observations? $\endgroup$ – tel Oct 13 '18 at 4:27
  • $\begingroup$ I don't know... :D I would assume whoever found identified a protein sequence would upload it in a shared data base and comparison among protein sequences (some ancestral equivalent to BLAST) allowed one to estimate the number of proteins found in humans. $\endgroup$ – Remi.b Oct 13 '18 at 5:19

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