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. Most translation occurs in the cytoplasm. Translation of secreted and transmembrane proteins occur at the surface of the endoplasmic reticulum. 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 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 complete set of chromosomes for a given specifies 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 and one circular mitochondrial chromosome. 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 all 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? 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

• Place each gene expression step in the correct order (RNA processing,transcription, translation)
  
• Where does RNA processing occur?
  
• Where does transcription occur?
  
• Where does translation occur?
  
• 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”. Genome coordinates are displayed along the X-axis. The annotations and graphics that describe gene structure, function, expression etc. are stacked along the Y-axis. 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 (24 linear chromosomes and the mitochondrial genome). Our focus will be on the human genome. Initially, we will focus our Genome Browser window on a single human gene, BBS1. In this chapter, 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”. Below that is a schematic of the chromosome that BBS1 is found on (Figure 1.5, G). The red vertical line (Figure 1.5, See asterix) indicates what part of the chromsome 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.

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), specific sequence variant (i.e. NM_024649.5:c.274T>C) or genome coordinates (i.e. chr2:157,736,446-157,876,330). Take some time to try all these navigational tools. To go back to BBS1, properly formatted, click once again on the BBS1 session link.

The BBS1 session link has been configured to contain only two 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 provides a graphical display of the genome coordinates. At this zoom level, you will only see a series of numbers separated by “|”. Those represent base position within the genome. Individual nucleotides will not be displayed unless you zoom in quote a bit. To quickly see individual nucleotides in the Base Position track click “zoom in” to base. To get back to where you were, click the back button of your web browser. The Gene Prediction track shows exon intron boundaries 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).

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 not 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

• What year was the most recent Human Genome Assembly uploaded to the UCSC genome browser?
  
• Go to the UCSC Genome Browser home page. Hover your mouse over the “Genome Browser” link in the toolbar at the top of the page. Choose “Reset all User Settings” in the pop up menu. Choose the Feb. 2009 Human Genome Assembly and click “Go”. How many evidence tracks are displayed in the Browser window?
  
• Where is BBS1 located on chromosome 11, the long arm or the short arm?
  
• Search for the gene, SOD1. Which chromosome is it on?
  
• Where is SOD1 located? On the long arm or short arm of the chromosome?
  
• Search for the gene, ACVR1. Which chromosome is it on?
  
• Where is ACVR1 located? On the long arm or short arm of the chromosome?
  
• Fill in the blank. Have you noticed a trend? The short arm of each chromosome is always positioned on the _______?
  
• First, use my session link to jump to BBS1. 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?

© 2019, Maria Gallegos. All rights reserved.