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johny 9:29pm on Sunday, September 5th, 2010 
At the time when I bought it (second half of 1998) the scanner was great value for money. Great scan quality, software is OK. This was our very first scanner. We were positively thrilled to have it, at first; and at that time we knew very little about computers.
LuvSeekerR 6:31am on Sunday, August 1st, 2010 
Great scan quality, software is OK, is recognized by Windows XP by default Not very fast, loud, big. scans are good quality poor compatibility, software conflicts, immense size, loud, slow, locks up system, poor durability

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doc0

Fermi National Accelerator Laboratory

FERMILAB-TM-2056

Digitizing Legacy Documents: A Knowledge-Base Preservation Project
Elizabeth Anderson, Robert Atkinson, Cynthia Crego, Jean Slisz and Sara Tompson
Fermi National Accelerator Laboratory P.O. Box 500, Batavia, Illinois 60510

September 1998

Operated by Universities Research Association Inc. under Contract No. DE-AC02-76CH03000 with the United States Department of Energy

Disclaimer

This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any speci c commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or re ect those of the United States Government or any agency thereof.

Distribution

Approved for public release; further dissemination unlimited.

Fermilab-TM-2056

by Elizabeth Anderson, Robert Atkinson, Cynthia Crego, Jean Slisz and Sara Tompson I. Introduction
The Fermilab Library was awarded a 1998 Educate and Automate grant from the Illinois State Library in the Digitizing Illinois Collections category.1 We applied for this grant in order to make our unique collection of scientific and technical reports publicly accessible and searchable on the Internet.
Since 1995, the Fermilab Library has been involved in the Technical Publications Fileserver Project. This project has made current scientific and technical publications available full text from our World Wide Web server and searchable via the Librarys online catalog. Our Web usage statistics (See Figure 1) demonstrate the significant use scientists, engineers, students and others make of this Internet-accessible knowledge base. Since the project began, we have gone from about 7,000 hits in 1995, to over 400,000 hits annually. (Which means approximately 40,000 documents are served to requestors each month.)
We have a large legacy collection of scientific and technical reports that we wanted to make publicly available. These reports, authored between 1972 and 1995, are held in paper format only. The legacy collection includes a large number of preprints, pre-publication versions of journal articles and conference presentations.
As more library customers and staff throughout the world come to rely upon rapid electronic access to fulltext documents, there is increasing demand to also make older documents electronically accessible. Illinois State Library grant funds allowed us to purchase hardware and software necessary to answer this demand. We created a production system to scan our legacy documents, convert them into Portable Document Format (PDF), save them to a server for World Wide Web access, and write them to CD discs for distribution. We hope our experience provides some guidance to others who are digitizing legacy collections.

Hardware and Software

Note: The authors are not endorsing any of the products mentioned in the following sections.
We already owned PC workstations and a UNIX server machine. Each workstation is a Micron Millennia Pro with a 200 Megahertz Pentium processor, a PCI 32-bit EIDE controller, 64 MB of memory (RAM), 2.1 GB EIDE hard drive, and a Micron 12x CD-ROM drive. The system board has 2 ISA slots and 4 PCI slots, 2 of which are taken by a Diamond Stealth video card and the network interface card.

The Fermilab reports are digitally archived on and served from our Web server machine, a SUN Sparc 20 with 256MB RAM, 2 1.05 GB hard drives plus an 8.4GB multidisk pack. This disk pack is configured under the Andrew File System (AFS). A number of physics laboratories share AFS space, which allows users to mount these volumes and use the documents as if they are locally accessible.

Hardware

We purchased the following hardware with grant funds: Two Microtek ScanMaker, Model E6, scanners, with Automatic Document Feeders Two Sony Spressa CSP-960S CD-recordable internal drives Two Adaptec 2910 SCSI Adapter Cards with 32-bit PCI bus (came with the Sony, but we also use for the scanner) TDK 74-minute recordable CD discs Micron Notebook PC
We purchased two scanners and CD-R drives so we could speed up production by having two people working at once, and purchased the notebook PC so we could set up mobile workstations for using Adobe Capture from our network. The Microtek ScanMaker E6 is a 30-bit, single-pass, flatbed scanner with an Automatic Document Feeder. The scanner is a SCSI device and came bundled with an ASPI-compliant SCSI interface card and the Microtek ScanWizard software. Scanners with a wide variety of features are available, but this less expensive scanner met our needs since we have same-sized documents and do not require high-resolution graphic output.
Since the SCSI host adapter card that came with the Microtek scanner was for an external device only, we used the Adaptec 2910 SCSI Adapter card for a PC workstation with a 32-bit PCI bus, which came with the SONY Spressa. Installing a SCSI host adapter is a necessity. Almost all CD-R drives are SCSI because SCSI provides the performance needed to "burn" a disc.2
CD recording or writing, known as CD-R, is rapidly becoming the medium of choice for storing scanned and digitized images of archival documents. 3 Others embarking upon this method of archival storage need to realize that writing to a CD-ROM is a CPU-intensive process. A recommended minimum configuration for the workstation to be used for CD-R is a 486DX2066 with 16MB RAM. 4
The Recordable CD-ROM drive we selected was the SONY Spressa CSP-960S that came bundled with the SONY CD Right software. We purchased an internal drive since CD-R drives are not considered as robust as floppy drives or CD-ROM drives. 5

TDK 74-minute, 650MB recordable discs were purchased for production. We chose the gold phthalocyanine discs rather than the green cyanine discs for archival purposes. The phthalocyanine dye is less sensitive to ordinary light and therefore will probably last longer and preserve information better. All phthalocyanine discs are supposed to last over 100 years.6 It is important to consider, however, that some CD players and CD-ROM drives will read data recorded on cyanine media more readily and reliably than they will read data recorded on phthalocyanine media.

Software

Microtek ScanWizard software came bundled with the scanner, and SONY CD Right software came bundled with the Sony Spressa drive. A large portion of the grant funding went for one software product, Adobe Acrobat Capture. Capture was purchased to convert the scanned output files, which are in TIFF (Tagged Image File Format) graphics format, to easily accessible PDF
(Portable Document Format) files. Capture allows us to scan documents and convert the scanned TIFF files to PDF format in one package. We purchased a version of Capture with a license that allows scanning of one million pages.

CD-R Installation

As is the case when setting up any computer system, it was important for us to pay attention in advance to the details of the configuration. We checked available expansion slots and bays in our workstations and determined the type of interface each device required, and made sure we had enough hard disk space to hold the data. In reviewing our configuration, we determined we needed an additional SCSI cable for each machine since the provided cables did not allow for one internal and one external device.
We began by installing the SCSI card into an available PCI slot and noting its ID, or address. The regular SCSI 2 system can handle 8 devices. Each device has to be assigned a unique ID, going from 0 to 7. The host adapter is considered a device itself and typically will occupy ID 7, although no actual standards exist to assign a particular ID to a certain type of device. In addition, the Interrupt Request (IRQ) address must be unique for each device. Duplication of SCSI IDs or IRQs can cause problems such as the device not being recognized by the host computer so it was vital that we made sure each device had a unique ID.
We installed the CD-R into an empty bay located below our existing CD-ROM drive and secured it with the provided mounting screws. We then connected the supplied ribbon cable from the CD-R to the SCSI host adapter card. We also connected a grounding wire that came with the

CD-R. We booted the system with the Easy-SCSI device-detection software provided with the Adaptec SCSI card to insure that both devices had unique IDs and were being recognized.
Next we attached a Mini-50 to Centron SCSI cable from the external port on the host adapter card to the Microtek scanner. We changed the SCSI ID on the scanner, using the SCSI ID wheel, which was external. Since the scanner was the last device in the SCSI "chain," we used a terminating resistor on the cable between the cable and the scanner port.

III. A.

Process Scanning Documents
The hardware and software detailed above is necessary for the legacy collection production process of scanning, converting, Web posting and CD-ROM writing. We began our scanning and conversion process by checking that all of our original paper documents were single-sided and clean with no marks, folds, creases, or bends. Necessary corrections were made with correction fluid and double-sided copies were copied single-sided on our copier.
The first step we took prior to scanning our first document was to set up in Capture both an input and an output folder for managing our documents during the scanning and conversion process. As noted above, Capture can be used for both scanning and converting. (See Figure 2, which illustrates both processes in the main Capture screen.) First, we created an input folder titled preprints by clicking on add input folder under the Input menu.
We chose the following settings for the input folder by right clicking on the preprints folder: Collate all Selected Files Save files in c:/capture2
Next, under the Output menu, we created an output folder titled preprints, and set the following settings by right-clicking on the preprints folder: File formats: Acrobat PDF [Image + Hidden Text] Processed Images: Retain in Place Suspect Settings: 95% Click all for ACD Under If Word Confidence. click PDF Under If Word Has. click PDF
Adobe Acrobat Capture gives the user the option to choose between three file formats for conversion to PDF: Acrobat PDF [Normal] Acrobat PDF [Image] Acrobat PDF [Image + Hidden Text]

After testing all three file formats, we found that the Image + Hidden Text format best suited our needs. Acrobat Capture will highlight words in both the Normal and Image formats that it does not recognize and save them as bitmap images in the PDF file. This often creates PDF files that contain bold highlighted words, increased font sizes from letter to letter in words, and
Digitizing Legacy Documents: A Knowledge-Base Preservation Project 7
characters and fonts not identical to the original. Documents created in one of these two formats needed additional proof reading and editing. We found this unacceptable. However, these two file formats did produce PDF files significantly smaller than those formatted in the Image + Hidden Text option. Since we were not concerned with file size, and since Image + Hidden Text results in an exact copy of the original paper document with no recognition suspects indicated, we chose it.
We are scanning each page of our documents using the following settings on our Microtek scanner: Type: line art Resolution: 300 dpi 8.5 x 11, 100%
To begin the scanning process, we load a document into the scanner document feeder and click the Scan button. We name each document by its Fermilab-assigned document number. Scanning is automatic, and when finished, all the files (TIFF format images) are organized in the Capture preprints input folder.
As an extra quality control step, we have been proofing each TIFF file in Adobe Photoshop. In our proofing process, we look for incomplete scans, any severe text shifts or missing pages. We have discovered that slightly slanted or crooked text in a TIFF document will automatically be straightened by Capture when converted to PDF. Note that screen resolution of TIFF images can be poor. However, the resolution improves once the pages are converted to PDF. If we see an
error with a TIFF file, we re-scan that page(s), renumbering it in the Capture scan filename dialog box. Renumbering is important, as it keeps the order of the pages intact. Note that we already owned Photoshop and use the software for other applications. If you dont own this software, TIFF files can be proofed using the Capture preview option.

Converting Documents

Back in Capture, we set the following preferences for converting the TIFF files to PDF by right clicking on the Process button: Performance Preference: Most Accurate Primary Language: English (US) Page Orientations: Portrait

We then select all TIFF files and click the Process button. We name the resulting PDF document using the same Fermilab-assigned document number. During the conversion process, the Processing Status lights appear. These indicate the processing steps as they occur.
When the conversion process is finished, the final PDF file appears in the preprints output folder. We then open the PDF file using Adobe Acrobat Exchange 3.0 and proof it against the original, looking for missing pages, missing text, etc. If we find an error, we then re-scan the page and run the document through the conversion process again. If we do not find an error, we transfer the PDF file over to our Webserver. Our department maintains a Web page with links to all of our reports at http://fnalpubs.fnal.gov/techpubs/pubs_lists.html. The reports are arranged
on this page in chronological order of date posted. We use the Library online catalog as the search engine for all reports.
We use Winsock FTP (a shareware program) to transfer the file from the hard drive on our PC workstation to the archive directory, our anonymous ftp area on our Webserver. We store all our documents in this area for retrieval by our customers. This directory, and thus all documents, are accessible via anonymous ftp, AFS space, our preprint Web pages, and the Fermilab Library online catalog.
We then add the URL for the PDF file to a basic HTML Web page. Each one of our reports has a corresponding HTML page with a link to the electronic file. Once the URL is added to the HTML page, the final step we take is to test that the link is working properly by viewing the page in our Web browser, currently Netscape 3.01.

CD-ROM Recording

Our first priority is posting the reports on the Web, but the grant support is also allowing us to prepare CD-ROMs each containing a full year of reports. The new CD-R technology makes CDROM writing a viable option for archival storage and information exchange.
A debilitating problem early on in the short history of CD recording was buffer underrun. CDROM drives have an internal data buffer that stores data being transferred to the CD-R disc. When the data can't get to the CD recorder fast enough to support the continuous data stream required, a buffer underrun may occur. Most so-called "third generation" recorders have

sufficient buffer sizes for most recording needs, including the SONY Spressa 960S. These new recorders create discs in ISO-9660 format so it should be readable in any computer equipped with a CD-ROM drive.
Copying the files to CD-R has proven to be surprisingly simple. Once the documents are scanned and placed on our server, we use ftp to put the files on our workstations hard drive. We launch the CD-Right software and click on the "Data CD" option from the main menu (See Figure 3).
From Windows NT Explorer, we then drag and drop (or cut and paste) the files to the "Recording List" area of the CD-Right window (See Figure 4.)
We give the volume a meaningful name and insert a blank CD-R disc. In a few seconds, the software determines whether the disc is blank or had data written to it from a previous session (See Figure 5.)
When the software indicates it is ready, we click on the Record button. We select the speed and then have to chose whether we want to Test, Test & Record if OK or just Record. We have decided to Test & Record. At a 4X speed, we are writing approximately 600 kilobytes per second. Keep in mind that a faster recording speed requires that the stream of data to the disc be continuous and therefore is more demanding of the CPU and hard drive.
The software begins recording, shows its status, and alerts one with sound and a notification window when finished. To Test & Record 100MB with our configuration takes less than 5 minutes.
The SONY Spressa 960s and the CD-Right software both support multisession, that is, the ability to record different sets of data to the same disc at different times, or sessions. Some older CDROM readers cannot read discs recorded in multiple sessions. To ensure maximum readability, we chose to copy all of our files to our hard disk, then write them all to the CD in one session. Again, our workstation configuration of a 2.1 GB hard drive made this possible.
Although our recording production has been running very smoothly, we have learned a few lessons: 1. Be sure to close all other applications while recording CDs. Memory-resident programs, such as calendars or screen savers may interfere with the recording process. 2. CD Right uses the Windows Temp directory, so make sure that directory has twice as much free space as the largest file you intend to record. 3. Keep the disc and the recorder free of dust.

4. CD-ROM Organization The TDK recordable media we purchased came with jewel boxes. We are purchasing preformatted CD-ROM label paper and will create labels for the boxes in-house. We will also include an insert with a contents note plus basic instructions on reading the CD. We will be placing a copy of Adobes free Acrobat Reader on each CD. Acrobat Reader is necessary for
viewing and printing the PDF format reports. The CDs will be readable by all Windows/Intelbased workstations.

Cataloging

The Fermilab Library began adding records for preprints and technical reports to our online catalog in 1993. The catalog is part of a Data Research Associates (DRA) integrated library system, which runs under VMS on a MicroVAX 3400 minicomputer.
In March 1994, the Library of Congress Network Development and MARC Standards Office promulgated a new edition of the USMARC Format for Bibliographic Data. In it was a specification for a new field, Electronic Location and Access, indicator 856.7 In June of that year, we began using this field to hold URLs in some of our catalog records. In June 1995, we began including URLs for electronic fulltext reports and preprints.
This paid off when, in late 1995, we purchased the DRA Web interface to our catalog. The 856 fields we had been adding became clickable hyperlinks from our Web catalog records directly to the resources represented by the records. It therefore became possible to find a record for a preprint or report in our catalog, click on its URL link, and view or print the complete document. Some free browser helper applications are necessary in order to view and print these documents, including Adobes Acrobat Reader for documents in PDF format, and the Ghostview Suite of applications for those documents in Postscript format (some of our reports are already on the Web in Postscript format).8 The ability to go right to the fulltext document from our catalog has became a routine function used daily by library customers and staff.
All Fermilab scientific and technical reports and preprints are fully searchable in the online catalog. The catalog is publicly available on the Web at: http://fnlib.fnal.gov/MARION. Extensive online help is available for the catalog at: http://wwwlib.fnal.gov/library/help/help.html.

Conclusion

A side benefit of digitizing the legacy document collection will be easier physical management of the documents. Currently we have five to six years of paper reports available in the Library for checkout. These stapled packets of paper are difficult to shelve and to keep in order, and take up many linear feet of bookcase ranges. Once we have all Fermilab reports available in two places, on the Web and on CD-ROM, we should be able to dispense with the paper copies, allowing customers to print the documents at the time they need them, rather than requiring the Library to store the reports just in case customers need them. We believe the PDF files on the CD-ROMs will be accessible for years to come, and represent a good format for archiving information. We will, however, retain one paper copy of each report, but only one.

Ultimately all Fermilab reports will be searchable and fulltext retrievable via the Fermilab Library Online Catalog. We anticipate having the documents back through 1990 available by the end of 1998, and earlier years will follow.
For those who do not have Internet access, the Fermilab Library can print, fax or e-mail documents to fulfill interlibrary loan or direct requests. We do not charge for interlibrary loans.
By the end of 1998, we will begin distributing a full years worth of Fermilab reports on CDROM for a minimal charge. The $20,000 Digitizing Illinois grant we received from the Illinois State Library is allowing us to make the entire body of scientific knowledge developed at Fermilab readily available to a broad audience.
Notes 1. FY98 Educate and Automate Grant in the Digitizing Illinois Collections category: Pioneering Science on the Energy Frontier: A Historic Knowledge-Base Preservation Project. Grant number 98-8015. 2. Miastkowski, Stan. Install a CD-Recordable Drive. PC World Online (October 1997). (On the Web at: http://www.pcworld.com/hardware/cd-rom_drives/articles/oct97/1510p356.html) 3. Parker, Dana J. and Robert A. Starrett. CD-ROM Professionals CD-Recordable Handbook: The Complete Guide to Practical Desktop CD. (Wilton, CT: Pemberton Press, 1996), p. 17. 4. Angus, Jeffrey Gordon and Carla Thornton. Do It Yourself CD-ROMs. PC World Online (January 1996). (On the Web at: http://www.pcworld.com/hardware/cdrom_drives/articles/jan96/jan9644.html) 5. Parker and Starrett, p. 91. 6. Parker and Starrett, p. 88. 7. The current specification is dated July 1997 and is in v.2 of the Format, which is available from the LC Cataloging Distribution Service. Related information is also available on the Web at: http://lcweb.loc.gov/marc/ 8. See the Librarys Web page at http://fnalpubs.fnal.gov/library/software.html for more information on helper applications.
The authors Elizabeth Anderson, Robert Atkinson, Cynthia Crego, Jean Slisz and Sara Tompson are all members of the Information Resources Department at Fermi National Accelerator Laboratory in Batavia, Illinois. Anderson is the Systems Librarian, Atkinson is the Collection Development Coordinator, Crego is the Department Manager, Slisz is the Technical Editor and Tompson is the Library Administrator.

doc1

Reprint requests to: Peter Sarnow, Department of Microbiology and Immunology, Stanford University School of Medicine, Fairchild Science Building, Stanford, California 94305, USA; e-mail: psarnow @leland+stanford+edu+
mRNA molecules at so-called internal ribosome entry sites (IRES) which are normally located hundreds of nucleotides downstream of the capped 59 ends of mRNAs+ Such IRES elements have been found in picornaviral (Jang et al+, 1988; Pelletier & Sonenberg, 1988; Jackson & Kaminski, 1995), flaviviral (TsukiyamaKohara et al+, 1992; Rijnbrand et al+, 1997), and cellular mRNAs (Macejak & Sarnow, 1991; Oh et al+, 1992; Teerink et al+, 1995; Vagner et al+, 1995; Gan & Rhoads, 1996; Bernstein et al+, 1997; Nanbru et al+, 1997)+ An RNA sequence element has been functionally designated as an IRES if it can mediate the translation of a second cistron in a heterologous dicistronic mRNA (Jackson & Kaminski, 1995)+ However, it is not clear whether all IRES elements identified in such dicistronic assays also function as IRESs in their normal locations in cellular mRNAs+ Therefore, a system was needed where the selective translation of normal cellular IREScontaining mRNAs could be monitored+ It has long been known that infection of cells with poliovirus results in the rapid inhibition of host cell translation, yet allows efficient translation of the viral mRNA
Cap-independent translation of eukaryotic mRNAs
(reviewed in Ehrenfeld, 1996)+ Several lines of evidence have shown that this translational inhibition is mostly due to an alteration of the cap-binding protein complex eIF4F+ For example, the eIF4G component of eIF4F is proteolyzed by viral (Liebig et al+, 1993; Haghighat et al+, 1996) and, perhaps, cellular proteases (Lloyd et al+, 1986; Wyckoff et al+, 1990) in infected cells+ Furthermore, the affinities of specific eIF4E-binding proteins for eIF4E are enhanced in poliovirus infected cells, resulting in a decreased affinity of eIF4E for eIF4G (Gingras et al+, 1996)+ Both the proteolysis of eIF4G and the sequestration of eIF4E by eIF4E-BPs lead to diminished levels of functional eIF4F complexes in infected cells+ As a consequence, translation of capdependent translation is severely inhibited (Etchison et al+, 1982)+ However, any cellular mRNA with a reduced requirement for eIF4F for translation initiation or which contains an IRES element, like polioviral mRNA, is predicted to be associated with host cell ribosomes and translated under such a condition+ In this study, we show that in addition to the immunoglobulin heavychain binding protein BiP mRNA (Sarnow, 1989), the natural IRES-containing c-myc and eIF4GI mRNAs were selectively associated with the translation apparatus during poliovirus infection, demonstrating that c-mycand eIF4GI-IRES elements function in their normal mRNAs and can confer selective translation to their respective mRNAs under stress conditions when overall cap-dependent translation is inhibited+

1502 Steady state levels of IRES-containing cellular mRNAs in 3NC202-infected cells First, the steady-state levels of certain mRNAs which lack or contain IRES elements, as determined by dicistronic assays, were determined at different times in 3NC202-infected cells+ Figure 2A shows that the steady state levels of non-IRES-containing actin (NakajimaIijima et al+, 1985) and junB (Nomura et al+, 1990) mRNAs remained constant throughout the infection+ Similarly, the levels of IRES-containing BiP (Sarnow et al+, 1986; Ting & Lee, 1988) and eIF4G (Yan et al+, 1992; Gan & Rhoads, 1996) mRNAs did not change significantly during the infectious cycle+ In contrast, IRES-containing c-myc (Arrushdi et al+, 1983; Bentley & Groudine, 1986; Nanbru et al+, 1997; Stoneley et al+, 1998) mRNA levels increased substantially at 3 h after infection, a time when viral 3NC202 mRNA molecules started to accumulate+ To determine whether the increase in c-myc mRNA level was due to the virusinduced inhibition of host cell translation, we monitored the steady-state levels of c-myc mRNA in uninfected cells in which translation was inhibited by addition of cycloheximide (Rose & Lodish, 1976)+ Specifically, cycloheximide was added to mock-infected cells at 1 h after infection and the steady-state levels of various mRNAs were examined at 0+5, 1, 2, 3, and 4 h after cycloheximide addition+ Figure 2B shows that treat-
G. Johannes and P. Sarnow
ment of cells with cycloheximide resulted in increased intracellular levels of c-myc at approximately 2 h after drug addition+ Therefore, the intracellular accumulation of c-myc mRNA is likely a consequence of virally induced inhibition of host cell mRNA translation+ This is in agreement with the observation that c-myc mRNA stability is controlled by a labile destabilizing factor (Dani et al+, 1984; Brewer & Ross, 1989)+
Association of natural IRES-containing mRNAs with the translation apparatus in infected cells IRES-containing mRNAs should be translated at 3 h after 3NC202 virus infection, because (1) cap-dependent translation is largely inhibited (Fig+ 1), (2) the intracellular levels of IRES-containing mRNAs are not diminished (Fig+ 2A), and (3) very little viral mRNA has accumulated at this time in the infected cells (Fig+ 2A), to compete with the cellular IRES-containing mRNA for the translational machinery+ To test the translation of cellular IRES-containing mRNAs, the polysomal distributions of IRES-containing and control mRNAs were examined in uninfected cells and in cells infected with 3NC202+ Figure 3 shows that all cellular mRNA species could be detected in polysomes which were isolated from uninfected cells (Mock)+ BiP (2,700 nt) and eIF4G

FIGURE 2. Steady-state levels of mRNAs in poliovirus infected cells+ A: Northern analysis of mRNAs during 3NC202 poliovirus infection+ Total RNA was isolated from mock-infected (M) or 3NC202-infected cells at various times after infection (16 h) and hybridized to radiolabeled DNA complementary to actin, Jun B, BiP, eIF4G, c-myc, or polioviral mRNAs+ An autoradiograph of the blot is shown+ The upper bands in the actin and eIF4G blots likely represent pre-mRNA species that disappear upon poliovirus infection because of the inhibition of polymerase II transcription (Crawford et al+, 1981)+ B: Effect of cycloheximide treatment on steady-state levels of mRNAs+ Total RNA was isolated from mock-infected HeLa cells at 0 h (lane 0) and 1 h (lane 1) prior to the addition of cycloheximide and at various times after cycloheximide additions (lanes 1+55)+ Addition of cycloheximide after 1 h is indicated by the arrow+ RNA isolated from control-treated cells (i+e+ no cycloheximide was added) taken in parallel with the 4-h time point is displayed in lane C+
FIGURE 3. Association of mRNAs with polysomes after inhibition of cap-dependent translation+ Polysomal distributions of mRNAs lacking (Actin, JunB) or containing (Bip, eIF4G, c-Myc, polio) IRES elements from mock- or 3NC202 poliovirusinfected cells at 2 h, 3 h and 4 h after infection are shown+ Absorbance profiles at 254 nm are shown at the top of each panel+ Fractions from the top to the bottom of the sucrose gradient are displayed from left to right+ Arrows denotes the migration of 60S ribosomal subunits+ Gradients were collected in 10 fractions+ Equal volumes of each fraction were loaded in each lane of the agarose gels+ The RNAs separated in the gels were transferred to membranes and hybridized to individually radiolabeled probes+ The autoradiograph was scanned using a Microtek ScanMaker E6 and the resulting image was labeled using Adobe Photoshop version 3+0+
1504 (5,100 nt) mRNAs were mostly associated with polysomal fraction 10, containing the most rapidly sedimenting polysomes+ Actin (1,900 nt) and c-myc (2,100 nt) mRNAs predominantly accumulated in polysomal fraction 9+ Both of these fractions are considered heavy polysome fractions, which contain mRNAs associated with more than seven ribosomes (Fig+ 3)+ In contrast, junB mRNA (1,800 nt) sedimented with polysomal fraction 8, indicating that junB mRNA was associated with fewer ribosomes on average, than was actin mRNA+ To test whether the sedimentation properties of these mRNAs were determined by the number of associated ribosomes, the sedimentation profiles of the mRNAs were examined after sucrose-gradient centrifugation in the presence of 15 mM EDTA, which causes the dissociation of ribosomes from mRNAs without disrupting the majority of nonribosomal RNAprotein complexes (Calzone et al+, 1982)+ Figure 4 shows that treatment with EDTA resulted in the disappearance of mRNAs from polysomal fractions 810, with concomitant accumulation of 40S and 60S ribosomal subunits+ These findings indicate that the presence of mRNAs in fractions 810 in gradients lacking EDTA was because of their association with ribosomes, and not with nonribosomal ribonucleoprotein complexes+

Inspection of Figure 3 shows that polysomes gradually disappear during the infectious cycle of poliovirus+ For example, approximately 50% of the polysomes redistributed into 40S and 60S ribosomal subunits by 2 h after infection+ Nearly 90% of polysomes were lost by 3 h after infection, and only small amounts of polysomes could be detected by 4 h after infection+ The disappearance of polysomes correlated well with the overall inhibition of host-cell mRNA translation shown in Figure 1+ Concomitant with the loss of polysomes, actin and junB mRNAs redistributed in the gradient from polysomal (Fig+ 3, Mock) to nonpolysomal fractions+ For example, the majority of both of these mRNAs redistributed from fractions 9 (actin) or 8 (junB) to fractions 16 by 3 h after infection+ Thus, the translation of actin and junB mRNAs is predicted to be severely inhibited (see below) as has been observed with most host-cell mRNAs at this time after infection (Fig+ 1)+ In contrast, the redistribution of BiP, eIF4G, and c-myc mRNAs was less dramatic, with much less than half of these three mRNAs sedimenting with nonpolysomal fractions 16 at 3 h after infection+ Table 1 displays quantitatively the distribution of the mRNAs+ Most of the BiP- (74%), eIF4G- (87%), and c-myc- (89%) IREScontaining mRNAs could be found in polysomal fractions 710 at 3 h after infection+ Although all mRNA species redistributed to the nonpolysomal fractions of the gradient by 4 h after infection, the IRES-containing mRNAs were still more quantitatively associated with polysomal fractions than the non-IRES-containing mRNAs (Table 1)+ Whether the preferential association of IRES-containing mRNAs with polysomes in infected cells resulted in their preferential translation during inhibition of cap-dependent translation was addressed in the next set of experiments+ Translation of non-IRES- and IRES-containing mRNAs in poliovirus-infected cells Extracts from cells that were radiolabeled with S 35 methionine for 10 min at different times after infection (Fig+ 1) were immunoprecipitated with antisera directed
TABLE 1+ Association of cellular mRNAs with polysomes in uninfected and poliovirus-infected cells+a
mRNA Mock 2Hr PI 3Hr PI 4Hr PI
FIGURE 4. Polysomal association of mRNAs in the presence of EDTA+ Polysomal analysis was carried out as described in Figure 3 (panel 3 Hr PI), except that 15 mM EDTA was included in the extraction and gradient buffer+ The absorbance profile at 254 nm is shown at the top of the panel+ The distribution of the various mRNAs within the gradient was monitored by Northern analysis as described in Figure 3+ The arrow denotes the migration of 60S ribosomal subunits+
Actin Jun B BiP c-Myc eIF4G
a Data are obtained after quantitation of the phosphorimage shown in Figure 3 using Imagequant software+ The relative amount of polysomal RNA found in polysomal fractions 710 compared to polysomal fractions in the Mock gradient (set as 100) is shown+

against actin, BiP, or c-myc proteins and analyzed in SDS-polyacrylamide gels followed by autoradiography+ Figure 5 shows that actin mRNA was translated in mocktreated cells (M) and at 1 h after infection (lane 1)+ Only a very small amount of actin was synthesized at 2 h after infection, and the synthesis of actin ceased completely by 3 h after infection+ This finding is in agreement with the observation that actin mRNA redistributed to nonpolysomal fractions by 3 h after infection (Fig+ 3), a time when overall cap-dependent translation was inhibited (Fig+ 1)+ In contrast, both newly synthesized BiP and c-myc proteins could be detected at 3 h after infection+ Therefore, the association of natural BiP and
1505 c-myc mRNAs with the translation apparatus in infected cells has functional consequences, that is, the continued synthesis of BiP (Sarnow, 1989) and c-myc proteins when overall cap-dependent translation is inhibited+ Unfortunately, several antibodies directed against eIF4G that have proven useful in the detection of eIF4G in Western blot analyses (Fig+ 1), were not able to immunoprecipitate radiolabeled eIF4G from uninfected cells (G+ Johannes & P+ Sarnow, unpubl+ observations); thus, the fate of newly synthesized eIF4G could not be examined by this approach+ Because eIF4G mRNA was clearly associated with polysomes in infected cells (Fig+ 3), we wanted to examine in more detail by which mechanism eIF4G mRNA could be translated in the absence of functional cap binding protein complex+ Isolation of a novel eIF4GI cDNA variant The eIF4G mRNA was detected in the above experiments with radiolabeled sequences that were complementary to the coding region of eIF4G mRNA, whose cDNA was isolated from a human brain cDNA library (Yan et al+, 1992)+ This eIF4G cDNA displayed an unusually long 59NCR, 368 nt in length (Fig+ 6A), which was subsequently shown to function as an IRES in dicistronic assays in cultured cells (Gan & Rhoads, 1996; Gan et al+, 1998)+ Curiously, radiolabeled sequences complementary to the 59NCR of eIF4G failed to hybridize to any mRNA species isolated from HeLa cells (G+ Johannes & P+ Sarnow, unpubl+ observations)+ Recently, Gradi et al+ (1998) published a novel cDNA variant of eIF4G, termed eIF4GI, which was identical to the originally isolated eIF4G cDNA except that it contained an extended N-terminus of 115 amino acids (Fig+ 6A)+ Because the identity between the previously isolated eIF4G and the eIF4GI cDNAs stopped abruptly at a putative canonical splice acceptor site (SA, Fig+ 6A), it was suggested by Gradi et al+ that the previously isolated eIF4G cDNA contained an unspliced intron at its 59 end that included the PPT and the IRES+ Thus, either the eIF4G and eIF4GI cDNAs represent spliced variants of the same RNA transcript, or they are derived from different genes or alleles+ To determine which variant eIF4G mRNAs were associated with polysomes in poliovirus-infected HeLa cells, we isolated cDNAs containing the 59NCRs of eIF4G genes from a human HeLa cDNA library (see Materials and Methods)+ Using PCR technology, several cDNAs were isolated that all contained at least the extra nucleotide extensions seen in eIF4GI (Fig+ 6A; Gradi et al+, 1998)+ While most of the isolated cDNAs contained the identical 59 terminal sequences found in the eIF4GI cDNA (Gradi et al+, 1998), several cDNAs contained longer 59 extended sequences+ Figure 6B shows the nucleotide sequence of the longest isolated cDNA, which added an additional 180 nt to the 59 end

methionine start codons at positions 196 and 199 (underlined in Fig+ 6B) each of which is embedded in optimal sequence context for translational initiation (Kozak, 1989)+ Two other AUG codons at positions 55 and 170
(underlined in Figs+ 6B and 6C) are not in optimal context+ However, because the entire sequence upstream of the AUGs at positions 196 and 199 encodes inframe protein sequence (Fig+ 6C), it is not known whether the authentic 59 end of the eIF4GIext gene was isolated+
1507 mRNA relocalized to nonpolysomal fractions (Fig+ 7)+ Similarly, radiolabeled probes that specifically hybridized to eIF4GIext mRNA displayed a polysomal distribution pattern of eIF4GIext mRNA that was identical to that of eIF4GI (G+ Johannes & P+ Sarnow, unpubl+ observations)+ Because the 59 ends of polysomal eIF4GI mRNAs have not yet been determined, it is not known whether eIF4GI mRNAs with different 59 ends are present in the cell+ In any case, these data suggest that eIF4GI mRNAs can be translated cap independently+
Polysome association of eIF4GI mRNAs in poliovirus-infected cells To determine whether the eIF4GI and eIF4GIext cDNA sequences were present in natural mRNAs, we analyzed the polysomal distributions of the eIF4GI and eIF4GIext mRNAs in uninfected and infected cells+ Using a radiolabeled probe (eIF4GI-59) that hybridized specifically to eIF4GI cDNA, the polysomal distribution of eIF4GI mRNA was examined in both mock- and virusinfected cells (Fig+ 7)+ The bulk of eIF4GI mRNA was associated with polysomal fraction 10 in uninfected cells (Fig+ 7, Mock), indicating that eIF4GI mRNA was efficiently translated in cells+ At 3 h after infection with 3NC202 poliovirus, the bulk of eIF4GI mRNA relocated to polysomal fraction 9, suggesting that eIF4GI mRNA was still translated at a time when the bulk of actin
Polysome association eIF4GII mRNAs in poliovirus-infected cells Recently, Gradi et al+ (1998) have identified a novel gene, eIF4GII, whose coding region shares an overall similarity of 56% with eIF4GI+ Importantly, eIF4GII seems to be a functional homolog of eIF4GI, because eIF4GII can restore cap-dependent translation to extracts in which eIF4GI has been proteolyzed (Gradi et al+, 1998)+ To monitor the polysomal association of the eIF4GII mRNA in uninfected and infected cells, cDNAs encoding the 59 NCR of eIF4GII were isolated from a HeLa

FIGURE 8. Test for internal ribosome entry using dicistronic mRNA expression+ A: Expression of dicistronic mRNAs containing various sequences in the intracistronic spacer (ICS) region+ In each transfection, observed luciferase activity was normalized to CAT activity+ In a typical experiment, CAT activity ranged from 520% of conversion of unmodified chloramphenicol to modified chloramphenicol+ Luciferase activity ranged from 5,00010,000 light units from EDcontaining constructs to 70,000100,000 light units for poliovirus IRES-containing constructs+ The results are displayed as luciferase light units/CAT units (normalized Luciferase/CAT) for two different experiments+ The dicistronic vectors contained ED- or ICS-control sequences (Macejak & Sarnow, 1991), the 59 terminal 325 sequences of the eIF4G-IIlong cDNA (G+ Johannes & P+ Sarnow, unpubl+ observations), the 59 terminal 196 sequences of the eIF4G-Iext cDNA, or the 59 terminal 747 sequences of the poliovirus cDNA in the intracistronic region+ Black and white boxes show results of two independent experiments each performed in triplicate transfections+ B: Integrity of dicistronic mRNA in transfected cells+ Poly(A ) mRNA was isolated from cells that were transfected with dicistronic plasmids, and analyzed by Northern blot hybridization using radiolabeled RNA that hybridized to chloramphenicol acetyltransferase (CAT) or luciferase (LUC) sequences+ The sequences located in the intercistronic spacer regions are indicated at the top of the figure+ The migration of 28S and 18S rRNA is indicated+ The autoradiograph was scanned using a Microtek ScanMaker E6 and the resulting image was labeled using Adobe Photoshop version 3+0+
bridized to the first CAT cistron or the second LUC cistron+ Figure 8B shows that the dicistronic mRNAs were largely intact, indicating that the translation of the second cistron was not mediated by a subgenomic mRNA+ Due to various transfection efficiencies, the intracellular steady-state levels of the different mRNAs changed from experiment to experiment (Yang & Sarnow, 1997)+ However, the expression of the first CAT cistron was always proportionate to the level of the mRNA+ For example, cell extracts prepared from dicistronic CATpolioLUC- or CAT 4GIIlongLUC-transfected cells displayed similar CAT activity (G+ Johannes & P+ Sarnow, unpubl+ observations)+ In summary, the extended 59NCRs in eIF4GI mRNA permitted internal initiation in the dicistronic assay and, thus, are likely to mediate the cap-independent translation of the natural eIF4GI mRNAs+ On the other hand, the extended 59 terminal sequences of eIF4GII did not function as an IRES in the dicistronic assay (Fig+ 8A)+ The failure to detect IRES activity in eIF4GII mRNA may indicate that the extreme 59 end of the mRNA has not yet been identified+ Alternatively, the observed capindependent translation of eIF4GII mRNA could be caused by a low requirement for the cap binding protein complex eIF4F+ DISCUSSION Although the list of IRES-containing cellular mRNAs is growing (Sachs et al+, 1997), it is often not clear whether such IRES elements function in their natural mRNAs+ This has been due to the lack of an experimental in vivo system in which cap-independent translation of IRES-containing mRNAs can be studied in the absence of ongoing cap-dependent translation of the bulk of the cellular mRNAs+ A logical system for studying cap-independent translation is poliovirus-infected cells in which cap-dependent translation is inhibited due to inactivation of eIF4F (Ehrenfeld, 1996)+ Using the mutant 3NC202 poliovirus that grows much more slowly than wild-type virus (Waggoner & Sarnow, 1998), it was possible to identify a time in the infectious cycle at which cap-dependent host-cell mRNA translation was inhibited, yet relatively low concentrations of viral mRNAs had accumulated+ The prediction that the cap-independent translation of natural IRES-containing mRNAs should be allowed under such circumstances was tested and it was found that certain IRES-containing mRNAs such as c-myc were not only associated with polysomes but also translated at 3 h in mutant virusinfected cells+ This finding shows that both the BiP IRES (Sarnow, 1989) and c-myc IRES function in their natural mRNAs+ It was found that eIF4G mRNA was translated cap independently in poliovirus-infected cells+ Surprisingly, the 59 end of the eIF4G cDNA, which we had isolated, had a different sequence than the 59 end of the eIF4G

1509 cDNA isolated by Yan et al+ (1992)+ In agreement with Gradi et al+ (1998), eIF4GIext mRNA, identified in human HeLa cells, contained the same coding sequences as eIF4G (Yan et al+, 1992) but a different 59 NCR+ These 59 end sequences in eIF4GIext functioned as an IRES in the dicistronic assay+ Thus, the cap-independent translation of eIF4GIext mRNA is likely to be conferred by the newly discovered IRES element+ The 59 NCR present in the originally isolated eIF4G cDNA (Yan et al+, 1992) clearly encodes a IRES (Gan & Rhoads, 1996; Gan et al+, 1998)+ However, the eIF4GIRES sequence is different from the eIF4GIext-IRES detected in this study+ The eIF4G-IRES contains a hallmark sequence that can be found in intron RNAs, that is, a polypyrimidine tract located upstream of the spliceacceptor sequence (Gan & Rhoads, 1996; Gan et al+, 1998)+ Whether the eIF4G-IRES encoding RNA species is a pre-mRNA or a functional mRNA that can be translated by ribosomes is not yet known+ If functional IRES elements are found in introns, it will be interesting to determine how such unspliced RNAs are translated, because introns are usually not exported from the nucleus into the cytoplasm (Chapon & Legrain, 1992; Hammarskjold, 1997)+ In this context, it is noteworthy that several IRES-binding proteins, such as La and PTB, are predominantly nuclear and are involved in RNAprocessing events+ Whether there is any interaction of these proteins with the nuclear export pathway is not known+ To date, we have no evidence for the mechanism by which eIF4GII can be translated cap-independently+ The main challenge will be to identify the mechanism that leads to cap-independent translation of mRNAs that are associated with polysomes in infected cells+ In addition to IRES-containing mRNAs, mRNAs with unstructured 59 NCRs that require only small amounts of functional eIF4F for their cap-dependent translation, such as heat shock mRNAs (Joshi-Barve et al+, 1992), may be identified this way+ Studies from Saccharomyces cerevisiae have indicated that the poly(A) binding protein (PABP), by interacting with either of the two eIF4G proteins, can greatly facilitate the recruitment of ribosomes to the 59 end of the mRNAs (Tarun & Sachs, 1995, 1996), indicating that the 59 and 39 ends in an mRNA can both regulate the efficiency of translation initiation+ Although an interaction of mammalian PABP and eIF4G proteins has not been observed so far, a human PABP-interacting protein (PAIP) that interacts with eIF4A has recently been identified (Craig et al+, 1998)+ Because eIF4A is known to be involved in several steps in translational initiation (Pause et al+, 1994), the PABPPAIP complex was suggested to provide a bridge between the 59 and 39 ends in mammalian mRNAs and perhaps promote reinitiation of ribosomes that had terminated at the 39 end (Craig et al+, 1998)+ If this model is correct, it is conceivable that mRNAs, which are translated by this presumably cap-inde-

1510 pendent reinitiation mechanism, are preferentially enriched in polysomes from infected cells+ The polysomal RNA pool from mutant-infected cells should be useful for several studies+ This pool should harbor several known and likely many unknown cellular mRNAs that can be translated cap independently+ Indeed, preliminary microarray hybridization experiments designed to detect mRNAs species that are preferentially associated with polysomes from infected cells have indicated that many hitherto unsuspected mRNAs can be translated cap independently (G+ Johannes, M+ Eisen, P+ Brown, & P+ Sarnow, unpubl+ observations)+ Several RNA viruses inhibit cap-dependent translation in infected cells+ This raises the question whether the translation products of cellular mRNAs that can be translated cap-independently have important functions in the viral infectious cycle and in viral pathogenesis+ MATERIALS AND METHODS Cells and virus
HeLa cells were maintained in Dulbecco modified Eagles medium (DMEM) supplemented with 10% calf serum containing 1 antimycotic and antibiotic solution (Gibco BRL)+ Mutant poliovirus, 3NC202, was used in all experiments (Sarnow et al+, 1986)+ Cells were infected with poliovirus at a multiplicity of infection of 30 as described (Sarnow et al+, 1986)+
Sal I and Nco I sites in the dicistronic vector (Macejak & Sarnow, 1991) to yield pSVCAT/4G-Iext /LUC and pSVCAT/ 4G-IIlong /LUC+
Transient plasmid transfections of cultured cells
HeLa cells were grown to 6070% confluency in 60 mm plates and transfected with 4 mg of plasmid DNA using 20 mg Lipofectin Reagent (Gibco BRL) per plate, following the manufacturers instructions+ Cells were harvested after incubation for 48 h at 37 8C and CAT and LUC activities were determined in the extracts as described (Ausubel et al+, 1989)+
Radiolabeling of cells and preparation of extracts
At various times after infection, 100-mm plates containing 6 cells were washed once with DMEM medium lacking methionine and cysteine+ Next, DMEM (meth, cys) containing 100 mCi/ml of 35 S methionine (3,000 Ci/mmol) was added and the plates incubated for 10 min at 37 8C+ The medium was removed and the cells washed twice with icecold PBS on ice+ The cells were scraped into 1 ml of ice-cold PBS, transferred to a 1+5-ml Eppendorf tube, and centrifuged at 1,000 rpm for 5 min at 4 8C+ The pellet was resuspended in ice-cold extraction buffer (50 mM Tris-Cl, pH 7+5, 5 mM EDTA, 150 mM NaCl, 1% NP40), vortexed briefly, and incubated on ice for 10 min+ Nuclei and debris were sedimented (10,000 rpm, 10 min) and the supernatant was saved+ Protein concentration was determined using the Biorad protein reagent (Biorad) with IgG as a standard+

for 12 h at 4 8C as described (Ausubel et al+, 1989)+ The membranes were incubated with 5% milk in Tris-buffered saline (TBS) overnight at 4 8C+ The blots were washed three times in TBS, incubated with polyclonal antibodies directed against eIF4G (Gradi et al+, 1998) (a gift from N+ Sonenberg, McGill University) for 1 h at room temperature, washed, incubated with horseradish peroxidase-conjugated anti-IgG, and visualized using the ECL kit following manufacturers directions (Amersham Life Sciences)+ Immunoprecipitations were performed with 20 mg of 35 S-methionine-labeled extracts using excess amount of antibodies directed against GRP78, myc, or actin (Santa Cruz Biotechnology) following the manufacturers procedure, except that the final wash solution included 500 mM NaCl+ The pellet was then resuspended in 30 ml of SDS-PAGE loading buffer and boiled for 10 min+ The beads were removed by short centrifugation and the supernatants loaded into the wells of a 10% SDS-PAGE gel+ The gel was dried and visualized after autoradiography+

Polysome analysis

Approximately 7 cells were mock infected or infected with 3NC202 poliovirus and incubated at 37 8C+ Prior to harvest, cycloheximide was added to the medium at a concentration of 0+1 mg/ml cycloheximide for 3 min at 37 8C + The medium was then removed and replaced with 10 ml of icecold PBS containing 0+1 mg/ml cycloheximide, and the plate was placed on ice+ All subsequent procedures where carried out on ice+ First, the cells were washed twice with ice-cold PBS containing 0+1 mg/ml cycloheximide and lysed directly on the plate after addition of 400 ml of polysome extraction buffer (15 mM Tris-Cl, pH 7+4, 15 mM MgCl2 , 0+3 M NaCl, 1% Triton X-100, 0+1 mg/ml cycloheximide and 1 mg/ml heparin)+ Extracts were transferred to 1+5 ml Eppendorf tubes and incubated on ice for 10 min with occasional mixing+ The nuclei and debris were removed by centrifugation at 12,000 g for 10 min in a microcentrifuge+ Supernatants were recovered and 500 ml aliquots were layered onto 10 ml, 1050% sucrose gradients composed of extraction buffer lacking Triton X-100+ The gradients were sedimented at 35,000 rpm for 190 min in a SW41 rotor at 4 8C+ The gradients were collected as 1+2 ml fractions by pumping 60% sucrose into the bottom of the gradient and collecting from the top using an ISCO fraction collection system, with concomitant measurement of the absorbance at 254 nm+ The fractions were adjusted to 2 ml by the addition of H2O, followed by the addition of 3 ml of 8 M guanidine HCL and vortexing extensively for 2 min+ Next, 5 ml of 100% ethanol was added and the fractions stored at 20 8C overnight+ The fractions were centrifuged at 10,000 rpm for 25 min to collect the RNA+ The pellets were washed with 75% ethanol, carefully resuspended in 400 ml of TE, pH 7+5, and precipitated again in 0+3 M NaOAC and 2+5 volumes of ethanol+ The pellets were washed with 75% ethanol and resuspended in 100 ml of TE, pH 7+5+ Equal volumes of each sample were analyzed by Northern analysis as described above+ All quantitations were performed using a Storm phosphorimager and Imagequant software (Molecular Dynamics)+ All analyses were performed three times and representative experiments are shown+ In the EDTA release experiment, MgCl2 was substituted with 15 mM EDTA in the extraction buffer and the gradient+

ACKNOWLEDGMENTS

We thank Karla Kirkegaard and Shelly Waggoner for critical reading of the manuscript+ This work was supported by NIH Grants R01 GM55979 (P+S+) and F32 GM16751 (G+J+)+ P+S+ was a recipient of a faculty research award from the American Cancer Society during the course of this study+
Received June 22, 1998; returned for revision July 23, 1998; revised manuscript received August 30, 1998

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Approximately 20 mg of total cell extracts (Waggoner & Sarnow, 1998) were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Biorad) at 25 V
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