RNA Secondary Structure as a Reusable Interface to ... - CiteSeerX

RNA Secondary Structure as a Reusable Interface to Biological Information Resources Ramon M. Felciano, Richard O. Chen & Russ B. Altman' Section on Medical Informatics Stanford University, MSOB X-215 Stanford, CA 94305-5479 USA {felciano, rchen, altman}@smi.stanford.edu 415-725-3394, (fax)415-725-7944 'To whom correspondences should be addressed. Keywords: RNA, domain graphic, user interface, component software, Java, secondary structure, 16S, 30S, Internet, World Wide Web Abstract The dissemination of biological information has become critically dependent on the Internet and World Wide Web (WWW), which enable distributed access to information in a platform independent manner. The mode of interaction between biologists and on-line information resources, however, has been mostly limited to simple interface technologies such has hypertext links, tables and forms. The introduction of platform-independent runtime environments facilitates the development of more sophisticated WWW-based user interfaces. Until recently, most such interfaces have been tightly coupled to the underlying computation engines, and not separated as reusable components. We believe that many subdisciplines of biology have intuitive and familiar graphical representations of knowledge that can serve as multipurpose user interface elements. We call such graphical idioms “domain graphics.” In order to illustrate the power of such graphics, we have built a reusable interface based on the standard two dimensional (2D) layout of RNA secondary structure. The interface can be used to represent any pre-computed layout of RNA, and takes as a parameters the sets of actions to be performed as a user interacts with the interface. It can provide to any associated application program information about the base, helix, or subsequence selected by the user. We show the versatility of this interface by using it as a special purpose interface to BLAST, Medline and the RNA MFOLD search/compute engines. These demonstrations are available at: http://wwwsmi.stanford.edu/projects/helix/pubs/gene-combis-96/.

Introduction With the explosion of biological sequence, structure and functional information, there have been major shifts in the way that data is published and used. The rise of the World Wide Web (WWW) in the last few years has created novel opportunities for publishing and sharing biological data. Among the most notable are efforts within the genome sequencing community. As sequences are deposited in the major databases, the curators of these collections have sensed the demand for integrated data retrieval tools, and have created a new generation of Internet-based tools for retrieval of bibliographic, sequential and other data[1, 2]. Sequence analysis tools are also now available on the Internet[3-5]. Other efforts at bringing data to the WWW include those that integrate the analysis of macromolecular structural data [6-8], metabolic pathways [9, 10] and the semantic connection between biological databases [11, 12]. These efforts have illustrated at least three important principles about the ways in which software support systems for biological research should be constructed. First, it has become clear that integrated systems offer the ability to move data from one phase of analysis to another in a

seamless manner, and greatly increase the quality (and fidelity) of the analysis. Second, these systems show us that general purpose interfaces, such as provided by the WWW facilitate the use of these services to a much greater degree than more platform-dependent specialized interfaces. For example, the ease in using the National Center for Biotechnology Information's (NCBI) BLAST server has made it the sequence homology search engine of choice for many investigators. Third, and most importantly, there is an immense potential for the construction of collaborative resources that allow complicated scientific reasoning tasks to be captured and semiautomated. Thus, distributed, platform-independent information resources with natural user interfaces are likely to be the most cost-effective and efficient way to bring biological data and computational tools to biologists. In this paper, we focus on the ways in which natural user interfaces can be identified and built in a reusable manner. Most WWW pages use standard Hypertext Markup Language (HTML)[13] forms and server-side Common Gateway Interface (CGI)[14] processes to allow access to on-line databases and other computational functionality. Often this is a form-based interface that requires (1) users to understand the query language of a particular database and (2) be able to express their questions using this query language. Such interfaces can be simplified by limiting the choices available to the user, but this comes at the expense of general utility, and is only appropriate for specific settings in which the interests/needs of the user can be fully anticipated. Platform-independent development technologies such as the Java language developed at Sun, Inc.[15] provide new opportunities for developing graphical interfaces to these same resources. In the context of the WWW, Java application code can be sent across the network to run on a wide variety of platforms. This allows for relatively efficient distribution of software modules, or “applets”, that can take advantage of local processing power without requiring manual installation by the user. Some work has focused on using Java as a platform-independent development environment to build stand alone biology applications, where the WWW is used to deliver the software to the client machine [16]. Others have used Java to add additional functionality to existing web-based applications [17, 18]. Few, however, have focused on the opportunity to build modular software components that can be used in multiple ways within a WWW page. There are commonly used graphics in biology which we will call domain graphics. They are widely used for printed communications, and they contain familiar symbols and layout patterns, but can be tailored for the problem being addressed. Thus, for example, RNA biologists may use a secondary structure graphic to highlight certain helices or mark bases of interest in an RNA sequence, but even without such markings, the fundamental meaning of a secondary structure graphic (as a representation of sequences, base pairs and partial structural information) is clear. A standard line graph or histogram, on the other hand, contains no domain knowledge: it is useless without lines or bars representing the data and a legend explaining the axes. Domain graphics are used throughout many scientific disciplines and can form the basis for powerful, intuitive and reusable user-interfaces. We have built a Java applet, SStructView, that interactively displays RNA secondary structures and links them to distributed network data resources, thus providing new interfaces to these resources. The power of the SStructView is in the simplicity of its parameter calls and action Uniform Resource Locator (URL)[19], which allow anyone with a working knowledge of HTML to include the SStructView applet in a WWW page and link it to other resources. SStructView does not have an algorithm for layout of RNA. Instead, it retrieves precomputed secondary structure layout files remotely as specified by a URL input parameter. SStructView then displays the secondary structure in the user’s WWW browser, and responds to user interaction by identifying which objects the user has clicked on. The selected objects can then be used to access other WWW resources by using them as parameters to search and compute engines. The applet supports several levels of zooming and uses offscreen imaging for optimized performance. The Java virtual machine architecture allows the SStructView to be run on a number of different platforms. In this paper, we describe the applet and show three examples of its use.

Materials and Methods SStructView is implemented as a Java applet using the Sun Java Development Kit 1.0[15]. The main classes defined in the code are Base, SecondaryStructure, SStructDisplayer, OffscreenImage and SStructView. In order to provide smooth and rapid scrolling, SStructView implements an offscreen image buffer. The offscreen image is rescaled to allow several levels of zooming on the secondary structure. SStructView has been tested on Macintosh, Windows NT, Silicon Graphics and Sun/Solaris systems, and can be viewed from any Java-capable WWW browser. All the data for the secondary structure is loaded when SStructView is launched. SStructView takes as input a two-dimensional description of the layout of the secondary structure (including information about sequence, base pairs and helical groupings), modeled after the NCSA imagemap file format[20]. Because this input takes the form of a URL, application developers can make their secondary structure algorithms available as WWW Common Gateway Interfaces (CGIs), and use the SStructView as an interface to view the results (Figure 1). The initial view presented by the applet is a “zoom out” view in which individual bases are represented as dots (Figure 2). As the user zooms in, the bases become letters (Figure 3). At any level of zoom, the mouse dynamically indicates the closest base. Single bases can be selected, as well as subsequences of bases, as described below (Figure 4). When a user makes a selection of a base or a sequence of bases, information about the selection is stored in selection variables which contain information about the base selected (type and sequence position, stored in variables base-id and base-val), any base pair of the selected base (type and position, pair-id and pair-val), the first and last base numbers selected if a sequence has been selected (subseq-start and subseq-end), and the actual sequence selected (subseq-val). All this information can be sent to an application program for subsequent processing. The applet takes four parameters following the HTML 3.2 specifications[13]: 1. structure-data-URL: the URL from which to obtain the secondary structure data in standard form. The secondary structure data file is a tab-delimited file where each line represents a base, base-pair, or helix. Each line contains a type tag indicating what type of biological structure is being defined, an identifier (ID) that is unique within the scope of the file, and a type-related parameter fields that provide the specific parameters for the associated type. The ID can be any alphanumeric set of characters. We use the base number as ID for bases, and short abbreviations such as “H1” for helices. The parameter fields are in order as follows. For bases, we specify: Base Letter (A, C, T or G), X-coordinate, Y-coordinate. For base pairs, we specify the ID of the two bases. For helices, we specify a comma-delimited list of base IDs. For example, the following describes several bases, four base pairs and a helix in the E. coli 16S secondary structure: BASE BASE BASE BASE BASE BASE ... BASE BASE BASE BASE BASE BASE

9 10 11 12 13 14

G A G U U U

201 205 209 212 216 220

259 258 257 256 255 258

20 21 22 23 24 25

U G G C U C

218 214 210 206 202 198

246 246 247 248 249 250

BASE BASE-PAIR BASE-PAIR BASE-PAIR BASE-PAIR HELIX

26 bp9 bp10 bp11 bp13 H1

A 195 251 9 25 10 24 11 23 13 21 9,10,11,12,13,21,22,23,24,25

2. action-URL: A URL to access when the user selects a base. The action-URL parameter is a URL that can take three forms. (1) It can be a standard URL which is invoked each time the user makes a selection, without regard to the selections the user has made. (2) If it ends with a “?” (the standard delimiter for query URLs), the interface appends all selection variable values to this URL before calling it, providing the application program with information about what base or subsequence of the RNA is of interest to the user. (3) If it contains a selection variable name bracketed by HTML comment markers (e.g. ““), then the brackets and sequence variable are replaced by the value of the variable (e.g. “GAUGG”). This provides a way for the application program to provide additional default information, while placing the selection information from the interface in the appropriate locations within the URL. 3. action-desc: A string used to describe the action-URL, that is used to label the button at the bottom of the applet. 4. result-display-frame: Specifies the Netscape frame in which to display the results of the action-URL. If this parameter is omitted or if the client browser does not support frames, the results will be displayed in the same window as the applet. The position of the bases for 16S ribosomal RNA, used in the examples shown below, were obtained by processing existing secondary structure graphics saved as postscript files. These files are available from the Ribosome secondary structure WWW Site [21, 22].

Results The SStructView interface follows a direct manipulation model: it presents data graphically to the user, allows the manipulation of that data with a mouse, facilities learning and retention, and encourages exploration of the data[23]. Users click on bases to select them. The selected base is marked in red; its base pair is marked in gray (both are labeled with their base number). If the base belongs to a helix, the other bases in the helix are highlighted as well. In addition, as the mouse moves over other bases, base numbers appear in order to allow browsing without removing focus from the selected base(s) or cluttering the display. Users select subsequences of the sequence by clicking on the start base and then shift-clicking on the end base. A set of magnification buttons allow users to zoom between four different zoom levels. Selections remain active as the user zooms. The applet performs some scale-sensitive “semantic zoom” as well: if the user zooms out far enough, only helix and other group data is shown. As the user zooms in, additional information is shown as the granularity increases and individual bases become visible. The user can scroll through large images by clicking on white space in the image and dragging. The use of a double buffering scheme allows scrolling to be relatively fast and smooth. We have found that scrollbars require too much screen space when the image is part of a larger HTML document. In order to illustrate the use of our interface applet, we have connected it to three

different information resources: BLAST, the MFOLD server, and an on-line version of Medline, used internally by the Stanford community.

BLAST The National Center for Biotechnology Information maintains a WWW accessible version of the BLAST sequence similarity and alignment engine[3]. Their interface provides a form in which biologists can enter a nucleotide sequence, and run a statistical comparison against similar entries in a number of nucleotide databases. We have used the sequence selection capabilities of SStructView as a graphical way to input this sequence data. Other BLAST search parameters are treated as constants for this application. The resulting application allows the biologist user to view a secondary structure, zoom in on a region of interest, select a sequence of bases from that region, and use BLAST to locate similar sequences. The applet code required to make a special purpose “16S-RNA-BLAST” application is shown here:

The structure-data-URL provides the information about the RNA sequence and layout of interest (for 16S RNA in this case). The action-URL contains the interface information necessary for interacting with the NCBI. Note that the action-URL parameter contains a reference to the subseq-val variable, which will be filled in by the SStructView before calling the BLAST server. An example of the output of this applet is shown in Figure 5. MFOLD Zuker et al have created an Internet accessible version of their mfold RNA folding prediction program[24, 25]. The server allows users to fold a sequence of bases by entering the sequence into a form. We have used SStructView as a graphical way to input the sequence data, using the 16S RNA layout. This allows biologists to interact directly with a familiar graphical image when entering data, and avoids typing errors. The entire applet code required for this “16S-RNAMFOLD” application is shown here:

An example of the output of this applet is shown in Figure 6. WebMedline

WebMedline is a prototype Internet-accessible application for searching the biomedical literature[26]. It is currently available only within local subnets. WebMedline takes as parameters keywords for a search, and then returns relevant articles. In the case of the 16S RNA, we can provide a number of standard keywords such as “16S” and “RNA” as constant, and provide selected base or helix as the third search criterion. Thus, a special purpose “16S-RNA-Medline” search application is shown here:

An example of the output of this applet is shown in Figure 7.

Discussion Using SStructView, we were able to quickly build task-specific interfaces using a common display metaphor. One could imagine an entire resource for RNA biology using this technology, and we are investigating the feasibility of building dynamically configured interfaces from domain graphicbased software components. Of course, the range of functionality in these three examples is limited, and we have not allowed users to vary the parameters sent to each of these engines. In some cases, this may be a valuable constraint to increase usability—especially when the context has been set by previous interaction with the user. However, in general, the limited ability to control the interface with the external information resource may be a source of frustration. SStructView uses an RNA layout that has been previously computed. Although there are numerous published layout algorithms, individual tastes differ sufficiently that the decision about which to use needs to be user-determined. In the case of 16S rRNA, we use a layout used widely by the community, and available over the Internet at the 16S Secondary Structural web site[22]. There is typically a tradeoff between user interface specificity and cost: developing graphically sophisticated interfaces tailored to a particular user community is more expensive than using conventional interface objects like buttons, checkboxes, imagemaps and dialog boxes. SStructView minimizes this tradeoff by providing researchers with a reusable, domain-specific interface object to use in their WWW interfaces that is faster and more effective than conventional interface objects. For example, to reproduce the functionality of SStructView using the imagemap and CGI standards, a static image of the secondary structure would be used as the primary focus of interaction. A click on a zoom button would call a CGI server process that would re-generate the image at a larger magnification and send it back to the browser, which would redisplay the entire page including the updated image. Such an interface would rely heavily on network bandwidth since the imagemaps would be regenerated after every interaction and be returned to the browser across the Internet. SStructView keeps the network communication to a minimum, and takes advantage of client-side processing power by keeping the image and secondary structure information local. Because of this performance increase, SStructView provides a directmanipulation interface to secondary structure information. Selection and display interactions occur directly with the graphical data, and auxiliary controls for zooming are kept to a minimum and are always available through a simple panel of controls. This move from speed-of-network to speedof-memory interactions provides a qualitative improvement over network user interfaces built with conventional WWW interface objects. And because the SStructView applet works with other

network-based components, development time and costs are minimal, and allow us to have 16S specific interfaces to BLAST, MFOLD, WebMedline and similar resources. Common Biological Knowledge Formats SStructView uses an ad hoc file format based on the NCSA Imagemap open standard. The lack of common knowledge interchange formats is a formidable barrier to building software systems out of third-party components. Muller et al have written a series of programs to generate similar files based on outputs from two-dimensional prediction programs[27]. Zuker’s MFOLD server produces a two-dimensional layout of the secondary structure which could theoretically be displayed by SStructView. Component architectures such as CORBA, OpenDoc and OLE provide methods for software components to specify their data structures and interfaces such that components can be combined to work together and share data[28]. For components like SStructView to be shared amongst researchers and coexist effectively with other components and software systems, there must be a common represention of this data. Weiser (personal communication) has proposed a data format for secondary structure data which could serve as the basis for such a standard. Components inside of WWW Browsers Software components that function in the context of WWW browsers are currently limited in their ability to interact with one another. Most plug-in modules are essentially self-contained application units that add functionality to the browser but do not interact with other components on the page. Once these limitations are overcome, applets like SStructView can coexist with other functional elements. For example, other interface components embedded in a WWW page will be able to set and retrieve selection and zoom settings from the SStructView applet. At this time, there are no standards for component interaction within a Web browser. SStructView is written as a reusable component, and not a standalone application. Helt and Rubin have developed a genome browser in Java that provides easy and timely access to genome data [16]. Their architecture differs from SStructView in that they use the Web primarily as a software delivery mechanism: though launched from a Web browser, the interface uses separate windows, dialog boxes, and menus, and is not linked to the contents of the Web browser. SStructView exists entirely inside of the Web browser window. Interactions occur inside the browser window as an integral part of an on-line HTML document. Applets that function inside of Web browsers are subject to the security conditions imposed by browser environment. Currently, other plug-ins or applications cannot manipulate SStructView to set or retrieve selection parameters. In addition, some browsers impose network related security restrictions that may limit which URLs can be called by SStructView. For example, the URLs that SStructView calls are limited to CGIs that support the GET protocol; Netscape 3.0b4 does not allow Java applets to send data to a host using the POST protocol. Thus, SStructView cannot be used as a front end for Web-based resources that only use the POST protocol. In the future, browsers may provide an methods for allowing applets and other plug-in components to communicate securely. Graphical User Interfaces in Biology Several other groups have developed systems that display RNA secondary structures. Muller, et al, created a system that displays secondary structures on a computer terminal and in PostScript formatted files [27]. Weiser and Noller have developed an interactive secondary structure viewer and editor, XRNA[29]. XRNA, written for systems supporting the X-Windows system, combines features of primary sequence analysis with secondary structural analysis and three-dimensional

analysis. While XRNA is primarily a display and editing tool, it shows the value of zooming and highlighting to focus attention when navigating the secondary structure. SStructView provides similar viewing and browsing functionality in component form and linked to on-line information resources. Searls, et al. have developed bioTk, a set of molecular biology Tk/TCL graphical interface objects[30]. Tk/TCL-based applications can run on most computers because TCL interpreters exist on a variety of platforms. These widgets allow application developers to create biology software sharing a consistent look and feel. Developers using the bioTk toolkit must be familiar with the Tk/TCL programming language, and must integrate these objects into their own source code. SStructView differs in that it requires only a familiarity with the HTML and URL open standards instead of a particular programming language. This allows SStructView to be integrated into “functional” WWW pages without source code level programming. SStructView is based on a custom two-dimensional display engine that supports zooming and panning (the basic graphics engine including with Java 1.0 does not support zooming and panning). The Virtual Reality Modeling Language 1.0 (VRML)[31] offers a WWW framework for supporting 3-dimension graphics with zooming and panning. However, VRML does not currently provide customizable support for multiple selections and highlighting, which limits its potential for building user interfaces. For example, it is difficult to highlight structures related to the one a user clicks on (a base pair or the helix to which a clicked base belongs). Some VRML browsers extend the standard WWW imagemap concept to allow clicking on a 3D structure to follow a URL. This functionality may be important for allowing 3D objects to be used as domain graphics, in the way that SStructView uses RNA secondary structure. VRML will need to support a wider variety of selection and response options. The secondary structure domain graphic is widely accepted by RNA biologists as an efficient and effective way of viewing sequence data and relationships. Schuster et al. have shown that secondary structure data can also be represented as an ordered rooted tree data structure[32]. This suggests that disciplines with isomorphic data representations can use similar display and interaction methods.

Conclusions The SStructView provides a familiar graphical representation of sequence data that can be used as a generic display and selection element embedded in a larger software tool. In particular, the SStructView is an interactive viewer for RNA secondary structure information designed to work in conjunction with other network-based software components and resources. It integrates twodimensional knowledge about the layout of the secondary structure (static or calculated at runtime) with a computational task through a familiar graphical representation. SStructView is easy to use and requires only network access to a file with the RNA secondary structural sequence and coordinates, and specification of where user-selection information (individual bases or subsequences of RNA) should be sent. SStructView can be viewed at http://wwwsmi.stanford.edu/projects/helix/pubs/gene-combis-96/.

Acknowledgments RMF and ROC are predoctoral fellows supported by LM-07033. RBA is a Culpeper Medical Scholar, and is supported by LM-05652 and LM-06422. Computing facilities were provided by the CAMIS Resource, LM-05305. The authors thank Bryn Weiser, Harry Noller and Tom Rindfleisch for useful discussions. The source code for SStructView is available from the authors upon request.

FIGURES

1. Client browser loads WWW page with embedded SStructView

Client WWW browser

2. SStructView retrieves secondary structure data from parameter URL.

HTTP or FTP server with base position data files.

3. User views secondary structure. and interacts with SStructView, selecting one or more bases. 4. User clicks the "action" button; SStructView calls the action-URL and displays the result in the browser window.

"Action" CGI or HTML page

Figure 1. Overview of a user session with SStructView. A client browser retrieves basic display data from a central source (far right), and allows the user to locally render and browse the secondary structure. When a selection is made and the user clicks the “action” button, the selected information is sent to the "action" CGI (bottom right) which performs the desired search or computation, and returns the result to the client. SStructView has been constructed so that the RNA specific information and the action CGI can be specified as parameters.

Figure 2. The overview level of display for SStructView. In this mode, the entire secondary structure is displayed showing only dots for individual bases and lines for basepairs. This offers a global view of the secondary structure, and allows the user to focus attention on areas of interest. As the mouse passes over individual bases, their base number (and the base number of the basepaired partner) is displayed, to orient the user.

Figure 3. At the medium view, SStructView provides the actual base types at each position in the sequence, as well as allowing mouse browsing dynamically and selection of individual bases.

Figure 4. SStructView in the Large view shows the individual bases, base pairs and helices at the highest resolution. As at all other levels, the user can select individual bases or subsequences. In this case, the user has selected base A33 and the system has highlighted both the base pair (U551) and the helix to which they both belong. The mouse has been moved over base A3 dynamically, but this base has not yet been selected.

Figure 5. Result of using SStructView with BLAST engine. The user has selected a subsequence of the E. coli 16S sequence, and pressed the button for BLAST search. SStructView has sent the sequence to BLAST with a standard set of parameters and the result of the search reveals a large set of matching sequences within GENBANK, including both E. coli and other organisms.

Figure 6. Result of using SStructView with MFOLD computational resource. The user has selected a subsequence of the E. coli 16S sequence, and pressed the button for MFOLD to compute an optimal fold. SStructView has sent the sequence to MFOLD with a standard set of parameters and the result is a report on the optimal local fold, the energetic parameters, and some display files for viewing the resulting secondary structure. MFOLD has folded the sequence into a similar configuration as displayed in the SStructView applet; a GIF image of this fold is available through the “.gif” link.

Figure 7. Result of using SStructView with WebMedline search engine. The user has selected a single base (C1409) from the E. coli 16S sequence, and pressed the button for MEDLINE search. SStructView has sent the individual base number, along with other elements of a search (including the identity of the RNA, 16S ribosomal RNA) to MEDLINE, and the result of the search reveals a set of Medline articles that specifically mention base 1409 either in the title or abstract.

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