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BACKGROUND: A new test (careHPV; QIAGEN, Gaithersburg, MD, USA) has been developed to detect 14 high-risk types of carcinogenic human papillomavirus (HPV) in about 2.5 h, to screen women in developing regions for cervical intraepithelial neoplasia (CIN). We did a cross-sectional study to assess the clinical accuracy of careHPV as a rapid screening test in two county hospitals in rural China. METHODS: From May 10 to June 15, 2007, the careHPV test was done locally by use of self-obtained vaginal and provider-obtained cervical specimens from a screening population-based set of 2530 women aged 30 to 54 years in Shanxi province, China. All women were assessed by visual inspection with acetic acid (VIA), Digene High-Risk HPV HC2 DNA Test (HC2), liquid-based cytology, and colposcopy with directed biopsy and endocervical curettage as necessary. In 2388 women with complete data, 441 women with negative colposcopy, but unsatisfactory or abnormal cytology or who were positive on HC2 or the new careHPV test, were recalled for a second colposcopy, four-quadrant cervical biopsies, and endocervical curettage. An absence of independence between the tests was not adjusted for and the Bonferroni correction was used for multiple comparisons. FINDINGS: Complete data were available for 2388 (94.4%) women. 70 women had CIN2+ (moderate or severe CIN or cancer), of whom 23 had CIN3+. By use of CIN2+ as the reference standard and area-under-the-curve analysis with a two-sided alpha error level of 0.0083, the sensitivities and specificities of the careHPV test for a cut-off ratio cut-point of 0.5 relative light units, were 90.0% (95% CI 83.0-97.0) and 84.2% (82.7-85.7), respectively, on cervical specimens, and 81.4% (72.3-90.5) and 82.4% (80.8-83.9), respectively, on vaginal specimens (areas under the curve not significantly different, p=0.0596), compared with 41.4% (29.9-53.0) and 94.5% (93.6-95.4) for VIA (areas under the curve significantly different, p=0.0001 and p=0.0031, for cervical and vaginal-specimen comparisons for the careHPV test, respectively). The sensitivity and specificity of HC2 for cervical specimens were 97.1% (93.2-100) and 85.6% (84.2-87.1), respectively (areas under the curve not significantly different from the careHPV test on cervical specimens, p=0.0163). INTERPRETATION: The careHPV test is promising as a primary screening method for cervical-cancer prevention in low-resource regions. FUNDING: Bill & Melinda Gates Foundation. [ABSTRACT FROM AUTHOR]

Radiation Biodosimetry is a continually developing clinical diagnostic field, which focuses on biological markers that proportionally change in relationship to the amount of ionizing radiation absorbed. Examples of host marker response include changes in white cell count, specific proteins in circulation, RNAs in white blood cells, or chromosome fidelity in affected lymphocytes. Measurements of radiation biomarkers correlate with the approximate radiation dose absorbed and indirectly provide an assessment of the likelihood of developing acute radiation syndrome. The aim of this review is to summarize four biodosimetry programs that are in advanced development, later pipeline stages with funding from the Biomedical Advanced Research and Development Authority (BARDA), an agency under the Assistant Secretary for Preparedness and Response (ASPR) in the U.S. Department of Health and Human Services (HHS). With BARDA financial support, biodosimetry diagnostic assays in development will inform patient management, improve health and psychosocial outcomes, and save lives after a nuclear disaster. These tests include an SRI International developed rapid on-site screening test requiring only a finger stick of blood to triage those who have received little or no radiation from those who have received clinically significant levels of radiation and need further immediate patient management. In addition, multiple laboratory-based, high-throughput quantitative tests, currently under development by MRIGlobal, DxTerity, and ASELL, will more accurately define dose levels and possibly predict cellular and organ-damage and other longer-term effects of radiation. In the future, when clinical and analytical validation of these assays is complete, the data is reviewed by the FDA, and agency use status is obtained, rapid triage and laboratory-based biodosimetry test results will enable emergency medical teams to do the most good for the largest number of people after a nuclear blast.

Background: There have been no published studies of carcinogenic human papillomavirus (HPV)--the necessary cause of cervical cancer--in Haiti, a nation that has one of the greatest burdens of cervical cancer globally., Objective: Characterize prevalence of carcinogenic HPV and the prevalence of individual carcinogenic HPV genotypes in women with cervical precancer or cancer, cervical intraepithelial neoplasia grade 2 (CIN2) or more severe (CIN2+)., Methods: Women (n=9,769; aged 25-60 years) were screened for carcinogenic HPV by Hybrid Capture 2 (HC2; Qiagen, Gaithersburg, MD). Carcinogenic HPV positives underwent colposcopy and visible lesions were biopsied. A subset of carcinogenic HPV positives was tested for individual HPV genotypes using a GP5+/6+ assay., Results: The prevalence of carcinogenic HPV was 19.0% (95% confidence interval: 18.4%-19.9%) and decreased with increasing age (ptrend < 0.001). Women with 3 or more sexual partners and who started sex before the age of 18 years had twice the age-adjusted prevalence of carcinogenic HPV of women with one partner and who started sex after the age of 21 (24.3% vs. 12.9%, respectively). HPV16 and HPV35 were the most common HPV genotypes detected in CIN2+ and more common in women with CIN2+ than those without CIN2+. HPV16 and/or HPV18 were detected in 21.0% of CIN2 (n = 42), 46.2% of CIN3 (n = 52), and 80% of cancers (n = 5)., Conclusions: The prevalence of carcinogenic HPV in Haiti was much greater than the prevalence in other Latin American countries. High carcinogenic HPV prevalence and a lack of cervical cancer screening may explain the high burden of cervical cancer in Haiti.

Success of the new human papillomavirus (HPV) DNA test for low-resource settings (careHPV™ test; QIAGEN Gaithersburg Inc., Gaithersburg, MD) requires good test performance when operated by personnel with limited laboratory experience. We evaluated the transferability, reliability, and accuracy of the careHPV test nested within a cervical screening project in a large Nigerian village. CareHPV testing was performed on screen-positive (n = 345) and screen-negative (n = 42) women attending colposcopy (68.3% of referred). Biopsies of abnormal-appearing areas were processed and read in the U.S. CareHPV specimens taken immediately before colposcopy were processed up to four times (in the field) by two secondary school graduates without laboratory experience, trained for this study. Specifically, QIAGEN Gaithersburg trained a laboratory-inexperienced U.S. researcher, who trained the first local technician who, in turn, trained the second. Residual specimens were sent to the U.S. for MY09/MY11 PCR testing for 13 carcinogenic genotypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) plus HPV66 (included in careHPV). Intrarater agreement was 98.8% (κ = 0.97) and 98.9% (κ = 0.97) for Technicians 1 and 2, respectively, while inter-rater agreement was 96.3% (κ = 0.90). Agreement with MY09/MY11 PCR (virologic reference standard) was 89.3% (κ = 0.73) with 74.2% sensitivity and 95.7% specificity. The careHPV test detected 12 (80%) of 15 histologically confirmed cervical intraepithelial neoplasia Grade 2 (CIN2) or worse lesions, with an estimated 83.0% specificity to detect

The analytic performance of a low-cost, research-stage DNA test for the most carcinogenic human papillomavirus (HPV) genotypes (HPV16, HPV18, and HPV45) in aggregate was evaluated among carcinogenic HPV-positive women, which might be used to decide who needs immediate colposcopy in low-resource settings ("triage test"). We found that HPV16/18/45 test agreed well with two DNA tests, a GP5+/6+ genotyping assay (Kappa = 0.77) and a quantitative PCR assay (at a cutpoint of 5000 viral copies) (Kappa = 0.87). DNA sequencing on a subset of 16 HPV16/18/45 positive and 16 HPV16/18/45 negative verified the analytic specificity of the research test. It is concluded that the HPV16/18/45 assay is a promising triage test with a minimum detection of approximately 5000 viral copies, the clinically relevant threshold., (Published by Elsevier B.V.)

Background: A next-generation diagnostic system has been developed at QIAGEN. The QIAensemble system consists of an analytical subsystem (JE2000) that utilizes a re-engineered Hybrid Capture chemistry (NextGen) to maintain the high level of clinical sensitivity established by the digene High-Risk HPV DNA Test (HC2), while creating improved analytical specificity as shown both in plasmid-based analyses and in processing of clinical specimens., Study Design: Limit-of-detection and cross-reactivity experiments were performed using plasmid DNA constructs containing multiple high-risk (HR) and low-risk (LR) HPV types. Cervical specimens collected into a novel specimen collection medium, DCM, were used to measure stability of specimens, as well as analytical specificity. Signal carryover, instrument precision, and specimen reproducibility were measured on the prototype JE2000 system using the automated NextGen assay., Results: The Limit of Detection (LOD) is <1000 copies of HPV 16 plasmid in the automated assay. No cross-reactivity (signal above cutoff) was detected on the automated system from any of 13 LR types tested at 10(7) copies per assay. Within-plate, plate-to-plate, and day-to-day performance in the prototype system yielded a CV of 20%. No indication of target carryover was found when samples containing up to 10(9) copies/ml of HPV DNA type 16 were processed on the JE2000 instrument. In an agreement study with HC2, 1038 donor cervical specimens were tested in both the manual NextGen assay and HC2 to evaluate agreement between the two tests. After eliminating discrepant specimens that were adjudicated by HR-HPV genotyping, the adjudicated positive agreement was 98.5% (95% CI: 94.6, 99.6)., Conclusions: The JE2000 prototype system automates NextGen assay processing, yielding accurate, reproducible, and highly specific results with both plasmid analytical model tests and cervical specimens collected in DCM. The final system will process more than 2000 specimens in an 8-hour shift, with fully continuous loading.

The diversity of higher-order structure in ribonucleoprotein (RNP) complexes makes them amenable to small-molecule modulation of their biological function. This review will discuss why bacterial RNase P, a simple yet essential ubiquitous and highly conserved RNP enzyme, represents an excellent target for the discovery and development of novel antimicrobials.

Iron-responsive elements (IREs) are the RNA stem loops that control cellular iron homeostasis by regulating ferritin translation and transferrin receptor mRNA stability. We mapped a novel iron-responsive element (IRE-Type II) within the 5'-untranslated region (5'-UTR) of the Alzheimer's amyloid precursor protein (APP) transcript (+51 to +94 from the 5'-cap site). The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain (+101 to +146). APP 5'-UTR conferred translation was selectively down-regulated in response to intracellular iron chelation using three separate reporter assays (chloramphenicol acetyltransferase, luciferase, and red fluorescent protein reflecting an inhibition of APP holoprotein translation in response to iron chelation. Iron influx reversed this inhibition. As an internal control to ensure specificity, a viral internal ribosome entry sequence was unresponsive to intracellular iron chelation with desferrioxamine. Using RNA mobility shift assays, the APP 5'-UTRs, encompassing the IRE, bind specifically to recombinant iron-regulatory proteins (IRP) and to IRP from neuroblastoma cell lysates. IRP binding to the APP 5'-UTR is reduced after treatment of cells with desferrioxamine and increased after interleukin-1 stimulation. IRP binding is abrogated when APP cRNA probe is mutated in the core IRE domain (Delta4 bases:Delta83AGAG86). Iron regulation of APP mRNA through the APP 5'-UTR points to a role for iron in the metabolism of APP and confirms that this RNA structure can be a target for the selection of small molecule drugs, such as desferrioxamine (Fe chelator) and clioquinol (Fe, Cu, and Zn chelator), which reduce Abeta peptide burden during Alzheimer's disease.

We performed a screen for drugs that specifically interact with the 5' untranslated region of the mRNA coding for the Alzheimer's Amyloid Precursor Protein (APP). Using a transfection based assay, in which APP 5'UTR sequences drive the translation of a downstream luciferase reporter gene, we have been screening for new therapeutic compounds that already have FDA approval and are pharmacologically and clinically well-characterized. Several classes of FDA-pre-approved drugs (16 hits) reduced APP 5'UTR-directed luciferase expression (> 95% inhibition of translation). The classes of drugs include known blockers of receptor ligand interactions, bacterial antibiotics, drugs involved in lipid metabolism, and metal chelators. These APP 5'UTR directed drugs exemplify a new strategy to identify RNA-directed agents to lower APP translation and A beta peptide output for Alzheimer's disease therapeutics.

In HeLa cells, ribonuclease P (RNase P), the tRNA processing enzyme consists of an RNA subunit (H1 RNA) associated with at least nine protein subunits, Rpp14, Rpp20, Rpp21, Rpp29 (hPop4), Rpp30, Rpp38, Rpp40, hPop1, and hPop5 (18.8 kDa). We report here the cloning and immuno-biochemical analysis of Rpp25, another protein subunit of RNase P. Polyclonal rabbit antibodies raised against recombinant Rpp25 recognize their corresponding antigens in RNase P-containing fractions purified from HeLa cells, and they also precipitate active holoenzyme. Furthermore, this protein has general RNA binding properties.

The inclusion of guanosine-5'-monothiophosphate (GMPS) in an in vitro transcription reaction facilitates enzymatic synthesis of an RNA transcript with a monothiophosphate group at the 5' end. A kinetic study of the modification reactions that generate monothiophosphate disulfide linkages with either 5'-GMPS alone or 5'-GMPS-primed RNA as the substrate revealed that the second-order rate constants increased as the pH was decreased. For example, when the reaction pH was lowered from 8 to 4, the k2 value for the coupling reaction between N-(6-[biotinamido]hexyl)-3'-(2'-pyridyldithio)propionamide (biotin-HPDP) and GMPS increased 67-fold from 1.84 to 123 M(-1) x s(-1). In addition to discussing a possible mechanism for coupling reactions that involve GMPS and disulfides, we also indicate conditions that are likely to be optimal for modification of the nucleophilic sulfur in 5'-GMPS-primed RNAs.

Background: Modern drug discovery is concerned with identification and validation of novel protein targets from among the 30,000 genes or more postulated to be present in the human genome. While protein-protein interactions may be central to many disease indications, it has been difficult to identify new chemical entities capable of regulating these interactions as either agonists or antagonists., Results: In this paper, we show that peptide complements (or surrogates) derived from highly diverse random phage display libraries can be used for the identification of the expected natural biological partners for protein and non-protein targets. Our examples include surrogates isolated against both an extracellular secreted protein (TNFbeta) and intracellular disease related mRNAs. In each case, surrogates binding to these targets were obtained and found to contain partner information embedded in their amino acid sequences. Furthermore, this information was able to identify the correct biological partners from large human genome databases by rapid and integrated computer based searches., Conclusions: Modified versions of these surrogates should provide agents capable of modifying the activity of these targets and enable one to study their involvement in specific biological processes as a means of target validation for downstream drug discovery.

RNA folds into complex structures that can interact specifically with effector proteins. These interactions are essential for various biological functions. In order to discover small molecules that can affect important RNA-protein complexes, a thorough analysis of the thermodynamics and kinetics of RNA-protein binding is required. This can facilitate the formulation of high-throughput screening strategies and the development of structure-activity relationships for compound leads. In addition to traditional methods, such as filter binding, gel mobility shift assay and various fluorescence techniques, newer methods such as surface plasmon resonance and mass spectrometry are being used for the study of RNA-protein interactions.

In HeLa cells, the tRNA processing enzyme ribonuclease P (RNase P) consists of an RNA molecule associated with at least eight protein subunits, hPop1, Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, and Rpp40. Five of these proteins (hPop1p, Rpp20, Rpp30, Rpp38, and Rpp40) have been partially characterized. Here we report on the cDNA cloning and immunobiochemical analysis of Rpp14 and Rpp29. Polyclonal rabbit antibodies raised against recombinant Rpp14 and Rpp29 recognize their corresponding antigens in HeLa cells and precipitate catalytically active RNase P. Rpp29 shows 23% identity with Pop4p, a subunit of yeast nuclear RNase P and the ribosomal RNA processing enzyme RNase MRP. Rpp14, by contrast, exhibits no significant homology to any known yeast gene. Thus, human RNase P differs in the details of its protein composition, and perhaps in the functions of some of these proteins, from the yeast enzyme.

At least six proteins co-purify with human ribonuclease P (RNase P), a tRNA processing ribonucleoprotein. Two of these proteins, Rpp30 and Rpp38, are Th autoantigens. Recombinant Rpp30 and Rpp38 are also recognized by Th sera from systemic sclerosis patients. Two of the other proteins associated with RNase P, Rpp20 and Rpp40, do not cross-react with Th sera. Polyclonal antibodies raised against all four recombinant proteins recognize the corresponding proteins associated with RNase P and precipitate active holoenzyme. Catalytically active RNase P holoenzyme can be separated from the nucleolar and mitochondrial RNA processing endoribonuclease, RNase MRP, even though these two enzymes may share some subunits.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an abundant nuclear protein that plays an important role in pre-mRNA processing and mRNA export from the nucleus. A1 shuttles rapidly between the nucleus and the cytoplasm, and a 38-amino acid domain, M9, serves as the bidirectional transport signal of A1. Recently, a 90-kD protein, transportin, was identified as the mediator of A1 nuclear import. In this study, we show that transportin mediates the nuclear import of additional hnRNP proteins, including hnRNP F. We have also isolated and sequenced a novel transportin homolog, transportin2, which may differ from transportin1 in its substrate specificity. Immunostaining shows that transportin1 is localized both in the cytoplasm and the nucleoplasm, and nuclear rim staining is also observed. The nuclear localization of A1 is dependent on ongoing RNA polymerase II transcription. Interestingly, a pyruvate kinase-M9 fusion, which normally localizes in the nucleus, also accumulates in the cytoplasm when RNA polymerase II is inhibited. Thus, M9 itself is a specific sensor for transcription-dependent nuclear transport. Transportin1-A1 complexes can be isolated from the cytoplasm and the nucleoplasm, but transportin1 is not detectable in hnRNP complexes. RanGTP causes dissociation of A1-transportin1 complexes in vitro. Thus, it is likely that after nuclear import, A1 dissociates from transportin1 by RanGTP and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing.

Protein import into the nucleus and export from the nucleus are signal-mediated processes that require energy. The nuclear transport process about which the most information is currently available is classical nuclear localization signal (NLS)-mediated nuclear import. However, details concerning the signal-mediated export of proteins and RNAs as well as alternative nuclear import pathways are beginning to emerge. An example of this is the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein which, by virtue of its M9 domain, is actively exported from the nucleus and imported into the nucleus via a novel pathway mediated by the recently characterized transportin protein. Here we report that the shuttling hnRNP K protein contains a novel shuttling domain (termed KNS) which has many of the characteristics of M9, in that it confers bi-directional transport across the nuclear envelope. KNS-mediated nuclear import is dependent on RNA polymerase II transcription, and we show that a classical NLS can override this effect. Furthermore, KNS accesses a separate import pathway from either classical NLSs or M9. This demonstrates the existence of a third protein import pathway into the nucleus and thereby defines a new type of nuclear import/export signal.

Human RNase P has been purified more than 2000-fold from HeLa cells. In addition to the RNA component, H1 RNA, polypeptides of molecular masses 14, 20, 25, 30, 38, and 40 kDa copurify with the enzyme activity. Sera from two different patients with the autoimmune disease scleroderma were used to immunodeplete human RNase P activity. These same sera cross-reacted on immunoblots with two of the copurifying polypeptides, p30 and p38, whereas an autoimmune serum that does not immunodeplete RNase P activity did not react with these proteins. Peptide fragments derived from purified p30 and p38 facilitated the molecular cloning and sequencing of cDNAs coding for these two polypeptides, which are now designated as Rpp30 and Rpp38, respectively. RPP38 cDNA encodes a polypeptide that may be identical to a previously identified antigen of approximately 40 kDa, which is immunoprecipitated by Th and To autoimmune antisera, and that has been implicated as a protein subunit of human RNase P by virtue of its ability to bind to H1 RNA in vitro. The second autoimmune antigen, Rpp30, as such, has not been described previously.

A simple strategy has been devised to identify the gene encoding the RNA subunit of RNase P from the zebrafish, Danio rerio. The sequence obtained by amplification of genomic DNA with primers based on sequences common to two other vertebrates was confirmed by reverse transcription and amplification of RNA from a partially purified preparation of the holoenzyme. The 5' and 3' ends were determined by cyclizing the RNA, followed by reverse transcription and sequencing across the ligated RNA junction. The zebrafish sequence is 63% identical to that of Xenopus laevis nuclear RNase P RNA and 69% identical to the human RNase P RNA. A consensus secondary structure was constructed based on these nucleotide identities and on the many compensatory base changes in several regions among these three RNAs. The strategy used to obtain the zebrafish sequence should be useful in deriving analogous gene sequences from diverse classes of eukaryotes.

Recently we have shown that the major isoform of RNase H in human cells, RNase H1, is able to cleave DNA substrates containing a single RNA-DNA base pair, an activity which appears to be involved in an excision repair system for the removal of ribose residues misincorporated into DNA. In the present work we have further characterized the substrate specificity of the enzyme. DNA substrates containing all four ribonucleotides are cleaved by the enzyme. A RNA-DNA base pair is not required for substrate recognition. RNA residues present within a mismatch or in a RNA-RNA base pair are also cleaved. The principal structural feature for recognition by the enzyme may simply be the presence of the 2'-OH group of the ribose residue adjacent to the cleavage site.

The pathways of degradation of oligodeoxynucleotides in plasma from several mammalian species, including human, were investigated. In all cases, hydrolysis occurred exclusively by a 3' to 5' exonucleolytic activity. Human, mouse, and rat plasma degraded oligonucleotides in this fashion at comparable rates, whereas rabbit plasma was severalfold more active. Single-stranded oligonucleotides were more susceptible to hydrolysis than double-stranded oligonucleotides. The rate of hydrolysis was sequence dependent: 3' pyrimidine nucleotides were cleaved more rapidly than 3' purines. The Km and Vmax values for an oligonucleotide 15-mer with the sequence TAGCACCATGGTTTC in human plasma were 50 microM and 4.5 microM/min, respectively. Substitution of the 3'-terminal phosphodiester internucleoside linkage with a phosphotriester rendered this substrate completely resistant to hydrolysis, showing that the enzyme is a pure 3' to 5' exonuclease and that there are no other nucleolytic activities in plasma. Modification at this position is required to inhibit rapid nuclease degradation of antisense compounds in vivo and in tissue culture systems requiring serum.

The major ribonuclease H from K562 human erythroleukemia cells has been purified more than 4,000-fold. This RNase H, now termed RNase H1, is an endoribonuclease whose products contain 5'-phosphoryl and 3'-hydroxyl termini. The enzyme has a native molecular weight of 89,000 based on its sedimentation and diffusion coefficients. Human RNase H1 has an absolute requirement for a divalent cation. Maximal activity is obtained with either 10 mM Mg2+, 5 mM Co2+, or 0.5 mM Mn2+. The pH optimum is between 8.0 and 8.5 in the presence of 10 mM Mg2+. The isoelectric point is 6.4. RNase H1 lacks double-stranded and single-stranded RNase and DNase activities, and it will not hydrolyze the DNA moiety of an RNA.DNA heteroduplex. Unlike the Escherichia coli enzyme, which requires a heteroduplex that contains at least four consecutive ribonucleotides for activity, human RNase H1 can hydrolyze a DNA.RNA.DNA/DNA heteroduplex that contains a single ribonucleotide. Cleavage occurs at the 5' phosphodiester of this residue. This substrate specificity suggests that human RNase H1 could play a role in ribonucleotide excision from genomic DNA during replication.

In Trypanosomatidae the messenger RNA's (mRNA's) that code for the variant surface glycoproteins (VSG's), tubulins, calmodulin, and at least a subset of other proteins contain a common 35-nucleotide leader sequence at their 5' ends. Hybrid-arrested in vitro translation has been used to show that all mRNA's in both African and South American trypanosomes contain this 35-nucleotide sequence. Oligonucleotides complementary to this sequence blocked translation of all trypanosome mRNA's in a rabbit reticulocyte lysate system, but did not inhibit translation of mRNA's from other organisms lacking this sequence. An oligonucleotide complementary to the VSG mRNA downstream from the spliced leader sequence arrested only VSG synthesis. Thus, the 35-nucleotide leader sequence is a general feature of all trypanosome mRNA's. The high specificity of oligonucleotides complementary to the spliced leader for their target sequence suggests that analogues permeable to the cell membrane may be useful in the treatment of trypanosomal infections.

The phosphorylation of protein 4.1 by the membrane kinase and casein kinase A has been investigated. Each of these kinases catalyzed the incorporation of 2 mol of phosphate per mole of protein 4.1. The presence of both kinases in the reaction mixture did not lead to an increase in the incorporation of phosphates into the protein. An analysis of the acid hydrolysis products of the 32P-labeled protein 4.1 indicated that the radioactivities were distributed between phosphothreonine and phosphoserine in a ratio of about 2 to 1. The effects of phosphorylation on the binding of protein 4.1 to spectrin were investigated by using sucrose density gradient centrifugation. The affinity of protein 4.1 for spectrin was reduced about 5-fold, from a KD of 2 X 10(-6) M to a KD of 9.4 X 10(-6) M, by phosphorylation. The phosphorylation of spectrin, on the other hand, appeared to increase slightly its affinity for protein 4.1. The results suggest that phosphorylation may lead to a relaxation of the cytoskeletal network and the formation of a more flexible membrane structure that is important to red cell function.