Evolution in an RNA world.

A long-standing research goal has been to develop a self-sustained chemical system that is capable of undergoing Darwinian evolution. The notion of primitive RNA-based life suggests that this goal might be achieved by constructing an RNA enzyme that catalyzes the replication of RNA molecules, including the RNA enzyme itself. This reaction was demonstrated recently in a cross-catalytic system involving two RNA enzymes that catalyze each other's synthesis from a total of four component substrates. The cross-replicating RNA enzymes undergo self-sustained exponential amplification at a constant temperature in the absence of proteins or other biological materials. Amplification occurs with a doubling time of approximately 1 hour and can be continued indefinitely. Small populations of cross-replicating RNA enzymes can be made to compete for limited resources within a common environment. The molecules reproduce with high fidelity but occasionally give rise to recombinants that also can replicate. Over the course of many "generations" of selective amplification, novel variants arise and grow to dominate the population based on their relative fitness under the chosen reaction conditions. This is the first example, outside of biology, of evolutionary adaptation in a molecular genetic system.

Circumstances at HIV diagnosis and progression of disease in older HIV-infected Americans.

OBJECTIVES: This study identified age-related differences in diagnosis and progression of HIV by analyzing a nationally representative sample of HIV-infected adults under care in the United States. METHODS: We compared older (> or = 50 years) and younger participants stratified by race/ethnicity. Regression models controlled for demographic, therapeutic, and clinical factors. RESULTS: Older non-Whites more often had HIV diagnosed when they were ill. Older and younger patients were clinically similar. At baseline, however, older non-Whites had fewer symptoms and were less likely to have AIDS, whereas at follow-up they had a trend toward lower survival. CONCLUSIONS: Later HIV diagnosis in non-Whites merits public health attention; clinical progression in this group requires further study.

The effect of cytidine on the structure and function of an RNA ligase ribozyme.

A cytidine-free ribozyme with RNA ligase activity was obtained by in vitro evolution, starting from a pool of random-sequence RNAs that contained only guanosine, adenosine, and uridine. This ribozyme contains 74 nt and catalyzes formation of a 3',5'-phosphodiester linkage with a catalytic rate of 0.016 min(-1). The RNA adopts a simple secondary structure based on a three-way junction motif, with ligation occurring at the end of a stem region located several nucleotides away from the junction. Cytidine was introduced to the cytidine-free ribozyme in a combinatorial fashion and additional rounds of in vitro evolution were carried out to allow the molecule to adapt to this added component. The resulting cytidine-containing ribozyme formed a 3',5' linkage with a catalytic rate of 0.32 min(-1). The improved rate of the cytidine-containing ribozyme was the result of 12 mutations, including seven added cytidines, that remodeled the internal bulge loops located adjacent to the three-way junction and stabilized the peripheral stem regions.

The impact of state policy on the costs of HIV infection.

There is substantial variation in the generosity of public assistance programs that affect HIV+ patients, and these differences should affect the economic outcomes associated with HIV infection. This article uses data from a nationally representative sample of HIV+ patients to assess how differences across states in Medicaid and AIDS Drug Assistance Programs (ADAP) affect costs and labor market outcomes for HIV+ patients in care in that state. Making ADAP programs more generous in terms of drug coverage would reduce per patient total monthly costs, mainly through a reduction in hospitalization costs. In contrast, expanding ADAP eligibility by increasing the income threshold would increase the total cost of care. Expanding eligibility for Medicaid through the medically needy program would increase per patient total costs, but full-time employment would increase and so would monthly earnings. The authors conclude that more generous state policies toward HIV+ patients--especially those designed to provide access to efficacious treatment--could improve the economic outcomes associated with HIV.

Medical care expenditures under gatekeeper and point-of-service arrangements.

OBJECTIVE: To compare expenditures for medical care in a closed-panel gatekeeper HMO and an open-panel point-of-service (POS) plan that share the same provider network. DATA SOURCE/STUDY SETTING: The two study HMOs are distinct product lines of a single managed care organization; both plans are commercial products. We used administrative data files from the study plans for 1994-95 to assess differences in total medical care expenditures and spending for five categories of services: physician services, inpatient hospital services, outpatient hospital services, prescription drugs, and other services. STUDY DESIGN: Multivariate analyses were based on the two-part model of the demand for medical care. The dependent variables in these models were expenditures in each of the five categories of services, and the independent variables were indicator variables for plan type and visit copayments, prescription drug copayment, distance to the nearest primary care physician (PCP), demographic characteristics, chronic conditions, area characteristics, and entry/exit indicator variables. PRINCIPAL FINDINGS: Total expenditures for medical care ranged from equal in both plans to 7 percent higher in the gatekeeper HMO (p < .10), depending on the copayments for physician visits. Expenditures were not higher in the POS plan for any of the five categories of services. These findings were robust to a wide range of sensitivity analyses. CONCLUSIONS: Direct patient access to specialists in POS plans does not necessarily result in higher medical care expenditures. When POS enrollees are required to choose PCPs, patient cost sharing, physician financial incentives, and utilization review may control expenditures without constraining direct patient access to providers.

RNA structure. Ribozyme evolution at the crossroads.

Molecules that have similar sequences usually adopt the same structures and have the same functions. In his Perspective, Joyce explains that this is not always the case. In a remarkable study (Schultes and Bartel), an RNA sequence has been designed that can adopt two different structures, each with a different catalytic function. Joyce details how this study sheds light on the evolution of enzymes.

A DNA enzyme with N-glycosylase activity.

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

Alternative conformations of a nucleic acid four-way junction.

A crystal structure of a 108 nucleotide RNA-DNA complex containing a four-way junction was solved at 3.1 A resolution. The structure of the junction differs substantially from the "stacked-X" conformation observed previously, due to a 135 degrees rotation of the branches. Comparison of the two conformers provides insight into the factors contributing to the flexibility of four-way junctions. The stacked-X conformation maximizes base-stacking but causes unfavorable repulsion between phosphate groups, whereas the 135 degrees -rotated "crossed" conformation minimizes electrostatic clashes at the expense of reduced base-stacking. Despite the large rotation of the branches, both junction structures exhibit an antiparallel arrangement of the continuous strands and opposite polarity of the crossover strands.

Expenditures for physician services under alternative models of managed care.

This study compares expenditures for physician services in a closed panel gatekeeper health maintenance organization (HMO) and an open panel point of service HMO that share the same physician network. The study uses administrative files of the two study HMOs for 1994-1995 to assess differences in spending for primary care physicians' (PCPs') services, specialists' services, and total physician services. When the copayments for PCP visits and PCP-referred specialist visits were $0, total physician expenditures were 4 percent higher in the gatekeeper HMO than in the point of service plan (p < .05). When the copayments for PCP visits and PCP-referred specialist visits were $10, total physician expenditures ranged from equal in both HMOs to 7 percent higher in the gatekeeper HMO (p < .01), depending on the copayment for self-referred visits. Expenditures for specialists' services were not higher in the point of service plan. The authors conclude that direct patient access to specialists does not necessarily result in higher physician or specialist expenditures in HMOs.

Visits to primary care physicians and to specialists under gatekeeper and point-of-service arrangements.

OBJECTIVE: To assess utilization of ambulatory visits to primary care physicians (PCPs) and to specialists in 2 different managed care models: a closed panel gatekeeper health maintenance organization (HMO) and an open panel point-of-service HMO. STUDY DESIGN: Retrospective study of patients enrolled in a single managed care organization with 2 distinct product lines: a gatekeeper HMO and a point-of-service HMO. Both plans shared the same physician network. PATIENTS AND METHODS: The study sample included 16,192 working-age members of the gatekeeper HMO and 36,819 working-age members of the point-of-service HMO. We estimated the number of PCP and specialist visits using negative binomial regression models and predicted the number of visits per year for each person under each HMO type and copayment option. RESULTS: There were more annual visits to PCPs and a greater number of total physician visits in the gatekeeper HMO than in the point-of-service plan. However, we did not observe higher rates of specialist visits in the point-of-service HMO. CONCLUSION: We found no evidence that direct patient access to specialists leads to higher rates of specialty visits in plans with modest cost-sharing arrangements.

RNA cleavage by a DNA enzyme with extended chemical functionality.

In vitro selection techniques were applied to the development of a DNA enzyme that contains three catalytically essential imidazole groups and catalyzes the cleavage of RNA substrates. Nucleic acid libraries for selection were constructed by polymerase-catalyzed incorporation of C5-imidazole-functionalized deoxyuridine in place of thymidine. Chemical synthesis was used to define a minimized catalytic domain composed of only 12 residues. The catalytic domain forms a compact hairpin structure that displays the three imidazole-containing residues. The enzyme can be made to cleave RNAs of almost any sequence by simple alteration of the two substrate-recognition domains that surround the catalytic domain. The enzyme operates with multiple turnover in the presence of micromolar concentrations of Zn2+, exhibiting saturation kinetics and a catalytic rate of >1 min-1. The imidazole-containing DNA enzyme, one of the smallest known nucleic acid enzymes, combines the substrate-recognition properties of nucleic acid enzymes and the chemical functionality of protein enzymes in a molecule that is small, yet versatile and catalytically efficient.

In vitro evolution of a ribozyme that contains 5-bromouridine.

The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.

A complex ligase ribozyme evolved in vitro from a group I ribozyme domain.

Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

A ribozyme that lacks cytidine.

The RNA-world hypothesis proposes that, before the advent of DNA and protein, life was based on RNA, with RNA serving as both the repository of genetic information and the chief agent of catalytic function. An argument against an RNA world is that the components of RNA lack the chemical diversity necessary to sustain life. Unlike proteins, which contain 20 different amino-acid subunits, nucleic acids are composed of only four subunits which have very similar chemical properties. Yet RNA is capable of a broad range of catalytic functions. Here we show that even three nucleic-acid subunits are sufficient to provide a substantial increase in the catalytic rate. Starting from a molecule that contained roughly equal proportions of all four nucleosides, we used in vitro evolution to obtain an RNA ligase ribozyme that lacks cytidine. This ribozyme folds into a defined structure and has a catalytic rate that is about 10(5)-fold faster than the uncatalysed rate of template-directed RNA ligation.

Crystallization of the 10-23 DNA enzyme using a combinatorial screen of paired oligonucleotides.

One of the most difficult steps in the X-ray crystallography of nucleic acids is obtaining crystals that diffract to high resolution. The choice of the nucleotide sequence has proven to be more important in producing high-quality crystals than the composition of the crystallization solution. This manuscript describes a systematic procedure for identifying the optimal sizes of a multi-stranded nucleic acid complex which provide high-quality crystals. This approach was used to crystallize the in vitro evolved 10-23 DNA enzyme complexed with its RNA substrate. In less than two months, 81 different enzyme-substrate complexes were generated by combinatorial mixing and annealing of complementary oligonucleotides which differed in length, resulting in duplexes of varying length, with or without nucleotide overhangs. Each of these complexes was screened against a standard set of 48 crystallization conditions and evaluated for crystal formation. The screen resulted in over 40 crystal forms, the best of which diffracted to 2.8 A resolution when exposed to a synchrotron X-ray source.

Variation in inpatient resource use in the treatment of HIV: do the privately insured receive more care?

OBJECTIVE: To estimate the impact of insurance status on inpatient resource use after adjusting for health upon admission and site of care. DESIGN: Detailed patient information linked to billing records from the AIDS Cost and Service Utilization Survey (ACSUS), a longitudinal analysis of inpatient and outpatient care between March 1991 and August 1992. SETTING: Hospitalizations of human immunodeficiency virus (HIV) patients from 10 US cities with high incidence of AIDS. PATIENTS: One thousand, nine hundred and forty nine adolescents and adults at various stages of HIV. MAIN OUTCOME MEASURES: We estimate inpatient charges, payments and length of stay as a function of patient, and provider and reimbursement characteristics for more than 1,500 hospitalizations to HIV patients. We control for patient characteristics and underlying risk factors including disease stage, CD4 percentage, mode of transmission, discharge status, type of admission, and region. We use hospital-fixed effects to control for unmeasured differences across facilities. RESULTS: Unadjusted means indicate that uninsured patients or patients covered by public insurance have significantly lower charges and payments than privately insured patients with similar medical conditions. We find that those differences are substantially reduced after controlling for the hospital in which care is received. Further, we find little evidence that "underinsured" patients are discharged sooner on average. CONCLUSIONS: Inpatient resource use is affected by both the hospital in which care is received and the type of patient admitted. Failure to control for unmeasured differences across hospitals is likely to overstate the impact of insurance substantially.

Crystal structure of an 82-nucleotide RNA-DNA complex formed by the 10-23 DNA enzyme.

The structure of a large nucleic acid complex formed by the 10-23 DNA enzyme bound to an RNA substrate was determined by X-ray diffraction at 3.0 A resolution. The 82-nucleotide complex contains two strands of DNA and two strands of RNA that form five double-helical domains. The spatial arrangement of these helices is maintained by two four-way junctions that exhibit extensive base-stacking interactions and sharp turns of the phosphodiester backbone stabilized by metal ions coordinated to nucleotides at these junctions. Although it is unlikely that the structure corresponds to the catalytically active conformation of the enzyme, it represents a novel nucleic acid fold with implications for the Holliday junction structure.