1 Genes, Genomes and Genome Browsers

1.1 What is a gene?

A gene is a segment of DNA that directs the production of a functional product (i.e. protein, see Figure 1.1). A significant number of eukaroytic genes are protein coding. Each protein-coding gene is first used as a template to create an RNA transcript through a process called transcription. The RNA transcript is then processed into a messenger RNA (mRNA). The most notable change that occurs during RNA processing is that intron sequences are removed (dark gray segments in Figure 1.1). The mRNA is then exported from the nucleus and used as a template to make a protein in a process called translation. All translation begins in the cytoplasm. Translation of secreted and transmembrane proteins continues at the surface of the endoplasmic reticulum (ER). These proteins do most of the work necessary to maintain cell structure, function and behavior.

Figure 1.1: An overview of Gene Expression. A small segment of DNA (light grey) containing a single gene (orange) is shown. The RNA transcript is highlighted in green (exons) and dark gray (introns).

While the transcribed region of a gene requires adjacent sequence to become transcribed at the right time and place (i.e. promoter, see Chapter 5), for simplicity, the size of a gene (number of base pairs) is typically defined by where transcription begins and ends. Thus, the sites of transcription initiation and transcription termination define both gene size and RNA transcript size (but not mRNA size which is typically shorter as most eukaryotic genes have introns).

1.2 What is a genome?

Genes are distributed linearly along chromosomes² (Figure 1.2). One unique set of chromosomes for a given species makes up what we call a genome (sometimes called the “haploid genome”)³.

Figure 1.2: This schematic represents a hypothetical segment of a chromosome with orange rectangles representing genes distributed linearly along the DNA segment shown.

The haploid genome for humans consists of 24 linear chromosomes (Figure 1.3) and one circular mitochondrial chromosome (not shown). Each chromosome varies in length. For example, chromosome one is the longest at 248,956,422 base pairs (bp). Given that 10 bp of a double helix measures 34 angstroms in length, chromosome one is calculated to be 82.7 mm!⁴ (Figure 1.3).

Figure 1.3: Actual length of the human genome when scale bar equals one centimeter. Naken mitochondrial DNA is too small to be shown. It is a small circular DNA molecule, with a circumference of 55 microns.

Like most animals, humans are diploid. Thus, their somatic cells⁵ harbor 22 pairs of autosomes⁶ and one pair of sex chromosomes (either XX or XY). Thus, over two meters of DNA is crammed into each somatic nucleus! But the diameter of the average mammalian nucleus is only 6 microns or 0.006 of a millimeter. How does the diploid genome fit? And how does it keep from becoming a tangled mess? First, the diameter of the DNA double helix is only two angstroms⁷ but also, each chromosome is carefully packaged by proteins in a systematic and stereotypical manner (Figure 1.4).

Figure 1.4: This image is modified from Uhler and Shivashankar, 2017. It shows the various levels of chromosome packing found in nuclei during interphase of the cell cycle. The first level of packing requires an octamer of histone proteins that assemble into a ball-like structure called a nucleosome. Naked DNA wraps around these octamers forming ‘beads on a string’. Additional levels of packing into chromatin fibers and topological associated domains requires additional proteins. Individual chromosomes (23 pairs in humans) are then carefully orgnanized within the cell nucleus in a cell type-dependent manner.

1.2.1 Test Your Understanding

• Order the gene expression steps (listed here in alphabetical order: RNA processing,transcription, translation)
  
• Indicate where each step occurs (listed here in alphabetical order:RNA processing,transcription, translation)
  
• TRUE or FALSE. If I told you the size of a mature mRNA transcript you would know the size of the gene in the genome?
  
• Fill in the blank. The first level of chromosomal packing requires an octamer of histone proteins that assemble into a ball-like structure called a _______
  

1.3 What is a Genome Browser?

According to Wikipedia, “Genome browsers enable researchers to visualize and browse entire genomes with annotated data including gene structure, protein structure, expression, variation, etc. They differ from ordinary biological databases in that they display data in a graphical format”. The genome sequence is displayed along the X-axis (when the sequence is not visible, coordinates are given). The annotations and graphics that describe gene structure, function, expression etc. are stacked below the genome sequence. Finally, the data used to create the graphics are contributed by multiple sources.

There are numerous Genome Browsers available. We will be using the UCSC Genome Browser. The UCSC Genome Browser harbors the sequence of sequenced genomes called “reference genomes”⁸ from a variety of species. Our initial focus will be on a single gene in the human genome, BBS1. In this chapter, using BBS1 as our example, you will learn how the UCSC Genome Browser is organized, how to configure so-called “evidence tracks”⁹ and how to navigate through Genome Browser window (scrolling left and right, zooming in and out).

1.4 Navigating the UCSC Genome Browser

To get started, click the BBS1 session link. You should be taken to the human BBS1 gene in the UCSC Genome Browser. Your web browser window will resemble Figure 1.5 but without the red annotations. Near the top of the page you will find the “Navigational Toolbar”(Figure 1.5, A-E). Below that is a schematic of chromosome eleven where BBS1 is located (Figure 1.5, G). The red vertical line within chromosome eleven (Figure 1.5, See asterix) indicates what part of the chromosome the browser window includes. Below that you will find the genome browser window centered on the human BBS1 gene (Figure 1.5, H/I).

Figure 1.5: An annotated version of the Genome Browser as it will appear when you click the saved session link provided in the text above. To move left or right see A. To zoom in or out, see B. To see where the browser window is located in the human genome, see C. This will indicate which chromosome BBS1 is on and the chromosomal coorindates. To see the size of the browser window, see D. To jump to a new location use the search window in E. G points to a graphical representation of the chromosome that includes BBS1. Below G, is the browser window itself. It consists of two evidence tracks, H and I. H points to the base position track and I points to the gene position track. J points to the name of the specific gene position track displayed. F points to an open triangle symbol that indicates that there is more to the ZDHHC24 gene that is shown in this window.

The BBS1 session link has been configured to contain only two so-called evidence tracks (see the two gray rectangles on the left side of the browser window to count): The Base Position track on top (Figure 1.5, H) and a Gene Prediction track on bottom (Figure 1.5, I). The Base Position track is a graphical display of genome position (or genome sequence when zoomed in to base level). It acts as a ruler but the units are in base pairs not inches. Like any ruler, you will see a series of numbers separated by vertical tick marks, “|”. Those represent position within the genome. At this zoom level, each tick mark represents a 1000 bp advancement (also see the scale bar at the top of the base position track, it should read 10 kb for kilobases). To view individual nucleotides in the Base Position track you will need to zoom in quite a bit or click “base” as your zoom in option. Try it! Notice that the scale of the ruler changes. To get back to where you were, click the back button of your web browser. The Gene Prediction track shows the position and length of each exon and intron for BBS1. Notice the gene name is displayed on the far left (Figure 1.5, BBS1 underlined). Also, notice that another gene (ZDHHC24) partially overlaps with BBS1! This is not that unusual. Also, notice that ZDHHC24 extends farther to the right as indicated by the presence of open triangles on the far right of the gene schematic (Figure 1.5, F).

You can use the navigational toolbar to scroll left <<< or right >>> (Figure 1.5, A) or zoom in or out (Figure 1.5, B). The length of DNA (in bp) displayed in the browser window is also indicated (Figure 1.5, D) including exact coordinates (Figure 1.5, E). Finally, there is a window that one can use to jump to a new gene (i.e. ACVR1). Try it. You will notice that when you start to type in the gene name (i.e. ACVR1), a drop down menu will appear. Click on the correct gene (more than one may be listed). This will take you directly to that gene in the genome browser (no need to click “go”). If instead you click “go” you will be taken to an intermediate page containing a long list of genome databases (We use “NCBI RefSeq genes”. This is less than ideal. To find your gene now, you might have to know something about where your gene is located (genome coordinates). You should go back and choose the proper gene from the drop down list.

You can also use this window to search for a specific sequence variant (i.e. NM_024649.5:c.274T>C) or genome coordinates (i.e. chr2:157,736,446-157,876,330). To go back to BBS1, properly formatted, click once again on the BBS1 session link.

The gray rectangles on the left side of the genome browser window (Figure 1.5, H and I) delineate the boundary of each evidence track displayed. Click on one of the gray rectangles and you will be taken to a “track settings” page for that particular evidence track. Try it. Explore the new page and the information it provides. Then click the back button to get back. You can also right click on a gray rectangle. This will open a small window for quick formatting. Quick formatting options include changing the way the information in the evidence track is displayed (choose hide, dense, squish, pack or full). You can also choose “View Image” to display a high resolution image of the browser window so that it can be downloaded for use in a presentation (NOTE: This knowledge will be useful for your Independent Project! - See the end of Chapter 8).

Popular evidence tracks are listed below the genome browser window. An advanced user might choose to show an evidence track from this list although any changes you make will not be displayed until you click the “Refresh” button (Figure 1.5, K). You can also see which evidence tracks are currently displayed (Figure 1.5, L and M). WARNING: The “UCSC Genes” evidence track will sometimes appear unexpectedly. This is a bug! When this happens the UCSC genes evidence track (Figure 1.5, N) will no longer be hidden. To minimize confusion, you should re-hide this track then click “Refresh”. It can be confusing when two gene prediction tracks are open at the same time.

Practice navigating through the genome browser. Zoom in. Zoom out. Scroll left. Scroll right. Click on a random position within the chromosome schematic. Hover your mouse over various elements in the browser window to read pop-up messages. Click on any feature for more details. Change track settings to see the impact. Then when you become hopelessly lost within the human genome or the formatting changes unexpectedly, use the BBS1 session link to get back.

The most important navigational trick to learn is how to “drag-and-select or click to zoom” from within the base position track. This trick will allow you to zoom in to a specific location within the browser window, a navigational trick we will use repeatedly. Master it now. Hover your mouse anywhere within the base position track until a tiny “drag select or click to zoom” popup message appears. Now you are in the right place to “click” then “drag” (with your finger on the trackpad) to perform the “Drag-and-Select” trick. When you do it correctly, you will create a box around the portion of the gene/genome you want to zoom into (red arrow, Figure 1.6) AND a “Drag-and-Select” window will open. Can’t get “click and drag” to work? Again, hover your mouse within the base position track until a tiny “drag select or click to zoom” popup message displays. Now you are in the right place to “right click” or “click” then drag to perform the “Drag-and-Select” trick.

Figure 1.6: The drag select or click to zoom tool is very useful.

Again, if you zoom in close enough you will see a short segment of the human genome sequence (top strand only) within the Base Position evidence track (Figure 1.7, A). How do I know the genome sequence displayed is the top strand¹⁰? Because the arrow to the left of the genome sequence (see red Asterix, Figure 2.2) is pointing to the right (—>). In molecular biology, the blunt end of an arrow represents the 5’ end of a DNA strand while the pointed end of an arrow represents the 3’ end. Always.

In the example shown in Figure 1.7, the Gene Prediction track displays an exon-intron junction (See asterix - intron is to the left, exon to the right). Moreover, the amino acid sequence is displayed on top of the exon schematic and positioned directly below each 3 bp codon. For example, “D” (aspartic acid) is coded by the G-A-C codon. Now you try it. Zoom into any exon-intron junction. If you do not see the amino acid sequence displayed on top of the exon schematic, right click on the gray rectangle corresponding to the Gene Prediction track then choose, “Configure RefSeq Curated”. Then change “Color track by codons:” from “OFF” to “genomic codons”. Take note, this trick may be useful in the future.

Figure 1.7: The drag-select tool was used to zoom in close to a small portion of an exon intron junction of BBS1. As you can see the first 4 nucleotides of the exon shown are G-A-C-C.

1.4.1 Test Your Understanding

• Use the search window to locate BBS1. HINT: When you type the gene name into the search window, a drop down window will appear. Click on BBS1 from the list and the window will automatically being to load a new window. DO NOT click “GO”. See your Manual for a more in depth explanation. Notice how the number of evidence tracks displayed increases by one (this is the bug). Which new gene prediction track has appeared?
  
• To reduce confusion, it is best to only have ONE gene prediction track open at a time. To close the duplicate gene prediction evidence track, right click on the grey rectangle at the left end of the window and choose “hide”. Or click on the grey rectangle corresponding to the duplicate gene prediction track and change the display mode to “hide”. Then hit Submit. Try this. If you have difficulties ask me or a classmate to show you how to do this.
  
• Search for the gene, SOD1. Which chromosome is it on?
  
• Find the scale bar in the base position track. How long is it (in kilobases, kb)?
  
• Find the genome ruler below the scale bar in the base position track, how many bp separate each vertical tick mark?
  
• Now use the session link I provide to find BBS1 again. Then use the Drag and Select tool to zoom into the beginning of exon 2 of BBS1. Continue to zoom in until you are close enough to see the genome sequence. What are the first four nucleotides?

© 2023, Maria Gallegos. All rights reserved.

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2. a chromosome is a single, long molecule of DNA↩︎

3. Many organisms are diploid (including humans) meaning they have two copies of each chromosome. Thus the phrase “haploid genome” is more precise, although the word “haploid” is often omitted and simply assumed↩︎

4. One way to calculate the length of a chromosome: Multiply the length of a chromosome in base pairs (bp) with 0.000000332, the length (in mm) of each bp.↩︎

5. Cells of a multicellular organism can be divided into two main types: germ cells and somatic cells. Germ cells are destined to become the reproductive cells like sperm and oocytes. Somatic cells are destined to become all the other cell types like skin, neurons and muscle. This distinction is made as somatic cells die with the death of the organism while germ cells have the potential to pass their DNA on to the next generation.↩︎

6. An autosome is one of the numbered chromosomes, as opposed to the sex chromosomes. Autosomes are numbered roughly in relation to their sizes. The largest autosome — chromosome 1 — has approximately 2,800 genes; the smallest autosome — chromosome 22 — has approximately 750 genes. This definition is found at https://www.genome.gov/genetics-glossary/Autosome↩︎

7. a unit of length equal to one hundred-millionth of a centimeter - Definition from Oxford Languages↩︎

8. A “reference genome” (also called a “reference assembly”) is a genome sequence created from thousands of sequence runs assembled in silico to represent the sequence of a genome of one idealized individual organism. Since it is assembled from sequence data obtained from a number of donors, reference genomes do not represent the sequence of any single individual or organism, but rather a mosaic of multiple donors↩︎

9. each evidence track harbors specific biological data from a single source pertaining to the sequence displayed in the browser window)↩︎

10. The top strand of DNA is also referred to as the “+ strand”↩︎