Chromatin three-dimensional interactions mediate genetic effects on gene expression.

Studying the genetic basis of gene expression and chromatin organization is key to characterizing the effect of genetic variability on the function and structure of the human genome. Here we unravel how genetic variation perturbs gene regulation using a dataset combining activity of regulatory elements, gene expression, and genetic variants across 317 individuals and two cell types. We show that variability in regulatory activity is structured at the intra- and interchromosomal levels within 12,583 cis-regulatory domains and 30 trans-regulatory hubs that highly reflect the local (that is, topologically associating domains) and global (that is, open and closed chromatin compartments) nuclear chromatin organization. These structures delimit cell type-specific regulatory networks that control gene expression and coexpression and mediate the genetic effects of cis- and trans-acting regulatory variants on genes.

Genetic characterization of CHO production host DG44 and derivative recombinant cell lines.

The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.

Use of air-cleaning devices to create airborne particle-free spaces intended to alleviate allergic rhinitis and asthma during sleep.

UNLABELLED: Comprehensive quantitative experiments were performed to assess the capabilities of several air-cleaning devices to create a particle-free microenvironment as a therapy for sleeping persons affected by allergic rhinitis and asthma. Six devices were evaluated, of which five were portable and intended to provide general air cleaning for bedroom-sized spaces. The sixth was intended for installation in front of the headboard of a bed and was designed to provide clean air focused in a space occupied by a sleeping person. The air-cleaning methods of the selected devices included high-efficiency particulate air (HEPA) filtering and electrostatic precipitation. Particle concentration measurements for six particle-size ranges and sound intensity measurements were made during 8-h, sleep-simulating periods. The effects of four parameters were studied: (i) device location, (ii) controlled air motion in the laboratory, (iii) airflow rate setting of the air-cleaning device, and (iv) controlled disturbances. To ensure a totally objective study, a special laboratory facility was constructed which enabled complete control of the experimental conditions. The measured concentration histories provided comprehensive evidence of the relative capabilities of the various devices for the specific air-cleaning function. It was found that the device designed to focus the cleaned air in the sleeping space fulfilled its goal and, in that regard, was clearly superior to all of the other air-cleaning devices. PRACTICAL IMPLICATIONS: There is evidence that allergic reactions are triggered by the presence of airborne particles and that these reactions can be mitigated by particle removal. This strategy can be implemented by the use of air-cleaning devices which are capable of creating particle-free zones at locations where human activity occurs. In particular, the creation of a particle-free zone which encompasses the breathing space of a sleeping person holds promise of mitigating sleep-disturbing allergic reactions.

Morphology and immunohistochemistry of spontaneous chronic corneal epithelial defects (SCCED) in dogs.

PURPOSE: To determine the morphologic features of the epithelium and extracellular matrix in spontaneous chronic corneal epithelial defects (SCCED) in dogs. METHODS: Forty-eight superficial keratectomy specimens were obtained after confirmation of the presence of a superficial corneal erosion for longer than 3 weeks with no discernible underlying cause. Histologic samples were examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. Immunolocalization of laminin, collagen IV, fibronectin, and collagen VII was performed. RESULTS: Epithelial cells adjacent to the defect were poorly attached to the underlying extracellular matrix. A prominent superficial stromal hyaline acellular zone composed of collagen fibrils in the area of the erosion was present in most specimens. Samples exhibited a varying degree of fibroplasia, vascularization, and leukocytic infiltrate. Laminin, collagen IV, and collagen VII were usually either not present or were present only in discontinuous segments on the surface of the erosion. Fibronectin usually coated the surface of the erosion, either as a continuous sheet or in discontinuous segments. Transmission electron microscopy of 15 samples revealed that the basement membrane was either absent in the area of the erosion or was present only in discontinuous segments. Scanning electron microscopy of eight of nine samples confirmed the absence of continuous basement membrane. Epithelial and extracellular matrix components in the peripheral cornea appeared normal. CONCLUSIONS: Most canine patients with spontaneous chronic corneal epithelial defects do not have a normal basement membrane structure in the region of the epithelial defect and have other abnormalities in the subjacent extracellular matrix that may reflect a part of the underlying pathophysiology of chronic and recurrent erosions.

In vitro analysis of an RNA binding site within the N-terminal 30 amino acids of the southern cowpea mosaic virus coat protein.

Southern cowpea mosaic virus (SCPMV) is a positive-sense RNA virus with T = 3 icosahedral symmetry. The coat protein (CP) has two domains, the random (R) domain and the shell (S) domain. The R domain is formed by the N-terminal 64 amino acids (aa) and is localized to the interior of the particle where it is expected to interact with the viral RNA. The R domain (aa 1--57) was expressed in Escherichia coli as a recombinant protein (rWTR) containing a nonviral C-terminal extension with two histidine tags. The RNA binding site of the R domain was identified by Northwestern blotting and electrophoretic mobility shift assay (EMSA) using recombinant wild-type and mutant R domain proteins. Deletions within the R domain revealed that the RNA binding site is localized to its N-terminal 30 aa. RNA binding by this element was found to be nonspecific with regard to RNA sequence and was sensitive to high salt concentrations, suggesting that electrostatic interactions are important for RNA binding by the R domain. The RNA binding site includes 11 basic residues, eight of which are located in the arginine-rich region between aa 22 and 30. It was demonstrated using alanine substitution mutants that the basic residues of the arginine-rich region but not those present at positions 3, 4, and 7 are necessary for RNA binding. None of the basic residues within the arginine-rich region are specifically required for RNA binding, but the overall charge of the N-terminal 30 aa is important. Proline substitution mutations within the N-terminal 30 aa, and alanine substitutions for prolines at positions 18, 20, and 21, did not affect the RNA binding activity of the R domain. However, it was demonstrated by circular dichroism (CD) that the conformation of the N-terminal 30 aa of the R domain changes from a random coil to an alpha-helix in the presence of 50% trifluoroethanol (TFE). The possible role for this structural change in RNA binding by the R domain is discussed.

Membrane activity of the southern cowpea mosaic virus coat protein: the role of basic amino acids, helix-forming potential, and lipid composition.

Southern cowpea mosaic virus (SCPMV) is a spherical RNA virus with T = 3 icosahedral symmetry. The particle is composed of 180 subunits of the coat protein (CP) and one copy of the positive-sense viral RNA. The CP has two domains, the random (R) domain formed by the N-terminal 64 aa and the shell (S) domain (aa 65--260). The R domain is highly charged, with 11 of the N-terminal 30 residues being basic. It is localized to the interior of the native particle where it may interact with the viral RNA, but under certain pH and salt conditions the topology of the particle changes to externalize the R domain. Since the CPs of several spherical RNA viruses have been shown to interact with host membranes during infection, we have begun investigating the membrane interactions of the SCPMV CP using the artificial liposome membranes. Both the native CP and the R domain overexpressed in Escherichia coli were observed to interact with liposomes. The interaction between the R domain and liposomes required either anionic phospholipids or non-bilayer-forming lipids and involved electrostatic interactions since it was shown to be both pH and ionic strength dependent. The analysis of four different deletion and six different site-directed substitution mutations partially mapped the region responsible for this interaction to residues 1--30. Analysis of this region of the R domain by circular dichroism indicated that it assumes an alpha-helical structure when exposed to liposomes composed of anionic lipids. Mutations, which extend the helical nature of this region, promoted an increased interaction. The possible role of the CP/lipid interaction in the SCPMV infection is discussed.

Breast cancer mortality after diagnostic radiography: findings from the U.S. Scoliosis Cohort Study.

STUDY DESIGN: A retrospective cohort study was conducted in 5573 female patients with scoliosis who were referred for treatment at 14 orthopedic medical centers in the United States. Patients were less than 20 years of age at diagnosis which occurred between 1912 and 1965. OBJECTIVES: To evaluate patterns in breast cancer mortality among women with scoliosis, with special emphasis on risk associated with diagnostic radiograph exposures. SUMMARY OF BACKGROUND DATA: A pilot study of 1030 women with scoliosis revealed a nearly twofold statistically significant increased risk for incident breast cancer. Although based on only 11 cases, findings were consistent with radiation as a causative factor. METHODS: Medical records were reviewed for information on personal characteristics and scoliosis history. Diagnostic radiograph exposures were tabulated based on review of radiographs, radiology reports in the medical records, radiograph jackets, and radiology log books. Radiation doses were estimated for individual examinations. The mortality rate of the cohort through January 1, 1997, was determined by using state and national vital statistics records and was compared with that of women in the general U. S. population. RESULTS: Nearly 138,000 radiographic examinations were recorded. The average number of examinations per patient was 24.7 (range, 0-618); mean estimated cumulative radiation dose to the breast was 10.8 cGy (range, 0-170). After excluding patients with missing information, 5466 patients were included in breast cancer mortality analyses. Their mean age at diagnosis was 10.6 years and average length of follow-up was 40.1 years. There were 77 breast cancer deaths observed compared with the 45.6 deaths expected on the basis of U.S. mortality rates (standardized mortality ratio [SMR] = 1.69; 95% confidence interval [CI] = 1.3-2.1). Risk increased significantly with increasing number of radiograph exposures and with cumulative radiation dose. The unadjusted excess relative risk per Gy was 5.4 (95% CI = 1.2-14.1); when analyses were restricted to patients who had undergone at least one radiographic examination, the risk estimate was 2.7 (95% CI = -0. 2-9.3). CONCLUSIONS: These data suggest that exposure to multiple diagnostic radiographic examinations during childhood and adolescence may increase the risk of breast cancer among women with scoliosis; however, potential confounding between radiation dose and severity of disease and thus with reproductive history may explain some of the increased risk observed.

Complementation of the host range restriction of southern cowpea mosaic virus in bean by southern bean mosaic virus.

Vigna unguiculata (cowpea) and Phaseolus vulgaris (common bean) are permissive hosts for southern cowpea mosaic virus (SCPMV) and southern bean mosaic virus (SBMV), respectively. Neither of these two sobemoviruses systemically infects the permissive host of the other. Although bean cells are permissive for SCPMV RNA synthesis, they do not support the assembly of this virus. Thus, the host range restriction of SCPMV in bean may occur at the level of movement and may involve the inability of SCPMV to assemble in this host. In this study, it was demonstrated that SCPMV accumulates in an encapsidated form in the inoculated and systemic leaves of bean plants following coinoculation with SBMV. No evidence was observed that the SCPMV that accumulated in coinoculated bean plants had an altered host range relative to wild-type SCPMV. These results suggested that SBMV complemented the host range restriction of SCPMV in bean. Additional experiments demonstrated that cowpea protoplasts are permissive for SBMV RNA synthesis and assembly. It was concluded from these results that the host range restriction of SBMV in cowpea occurs at the level of movement. In mixed infections of cowpea with SCPMV and SBMV, the latter was recovered from the inoculated but not the systemic leaves. Its recovery from the inoculated leaves, however, was not dependent on the presence of SCPMV in the inoculum. From these results, it was concluded that SCPMV did not complement the host range restriction of SBMV in cowpea.

The 105-kDa polyprotein of southern bean mosaic virus is translated by scanning ribosomes.

The cowpea strain of southern bean mosaic virus (SBMV-C) is a positive-sense RNA virus. Three open reading frames (ORF-1, ORF2, and ORF3) are expressed from the genomic RNA. The ORF1 and ORF2 initiation codons are located at nucleotide (nt) positions 49 and 570, respectively. ORF1 is expressed by a 5' end-dependent scanning mechanism, but it is not known how ribosomes gain access to the ORF2 initiation codon. In experiments described here, it was demonstrated that the translation of ORF2 was sensitive to cap analog in a cell-free extract. In vitro and in vivo studies showed that the addition of one or more AUG codons between the 5' end of the SBMV-C RNA and the ORF2 initiation codon reduced ORF2 expression and that elimination of the ORF1 initiation codon increased ORF2 expression. Altering the sequence context of the ORF1 initiation codon to one more favorable for translation initiation also reduced ORF2 expression in vivo. Nucleotide deletions and insertions between SBMV-C nt 218-520 did not abolish ORF2 expression. In most cases, these mutations resulted in reduced expression of both ORF1 and ORF2. These results are consistent with translation of ORF2 by leaky scanning.

Identification of viral genes required for cell-to-cell movement of southern bean mosaic virus.

Inoculation of Vigna unguiculata (cowpea) with transcripts synthesized in vitro from a genome-length cDNA clone of the cowpea strain of southern bean mosaic virus (SBMV-C) resulted in a systemic SBMV-C infection of this host. Capped RNA was about five times more infectious than uncapped RNA as determined by a local lesion assay. The SBMV-C cDNA clone was also used for mutagenesis of the four SBMV-C open reading frames (ORFs). ORF1, ORF3, and coat protein (CP) mutants were not infectious in cowpea. Electroporation of cowpea protoplasts with mutant transcripts demonstrated that the ORF1, ORF3, and CP gene products were not required for SBMV-C RNA synthesis, and the ORF1 and ORF3 gene products were not required for SBMV-C assembly. From these results, it was concluded that the ORF1 and ORF3 proteins and the CP are required for SBMV-C cell-to-cell movement. One of the ORF3 mutants pSBMV2-UAA1833 contained a nonsense codon between the predicted -1 ribosomal frameshift site (SBMV-C nucleotides 1796-1802) and a potential ORF3 translation initiation codon at SBMV-C nucleotide 1895. The lack of infectivity of this mutant suggested that ORF3 was expressed by a -1 ribosomal frameshift in ORF2 rather than by initiation of translation at nucleotide 1895.

Mapping and expression of southern bean mosaic virus genomic and subgenomic RNAs.

The coat protein of the cowpea strain of southern bean mosaic sobemovirus (SBMV-C) is translated from a subgenomic RNA (sgRNA) that is synthesized in the virus-infected cell. Like the SBMV-C genomic RNA, the sgRNA has a viral protein (VPg) covalently bound to its 5' end. The mechanism(s) by which ribosomes initiate translation on the SBMV-C RNAs is not known. To begin to characterize the translation of the sgRNA it was first necessary to precisely map its 5' end. Primer extension was used to identify SBMV-C nucleotide (nt) 3241 as the transcription start site. As a control, the 5' end of the genomic RNA was also mapped. Surprisingly, the 5' terminal nt of this RNA was identified as SBMV-C nt 2. The primary structure of the 5' ends of these two RNAs is therefore expected to be VPg-ACAAAA. Precise mapping of the 5' end of the sgRNA of the bean strain of SBMV (SBMV-B) demonstrated that it has these same elements. Translation of coat protein from the SBMV-C sgRNA and p21 from the SBMV-C genomic RNA was compared using a cell-free system. The results of these experiments were consistent with translation of these proteins by a 5' end-dependent scanning mechanism rather than by internal ribosome binding.

Determination and comparative analysis of the small RNA genomic sequences of California encephalitis, Jamestown Canyon, Jerry Slough, Melao, Keystone and Trivittatus viruses (Bunyaviridae, genus Bunyavirus, California serogroup).

The nucleotide sequences of the small (S) genomic RNAs of six California (CAL) serogroup bunyaviruses (Bunyaviridae: genus Bunyavirus) were determined. The S RNAs of two California encephalitis virus strains, two Jamestown Canyon virus strains, Jerry Slough virus, Melao virus, Keystone virus and Trivittatus virus contained the overlapping nucleocapsid (N) and non-structural (NSs) protein open reading frames (ORFs) as described previously for the S RNAs of other CAL serogroup viruses. All N protein ORFs were 708 nucleotides in length and encoded a putative 235 amino acid gene product. The NSs ORFs were found to be of two lengths, 279 and 294 nucleotides, which potentially encode 92 and 97 amino acid proteins, respectively. The complementary termini and a purine-rich sequence in the 3' non-coding region (genome-complementary sense) were highly conserved amongst CAL serogroup bunyavirus S RNAs. Phylogenetic analyses of N ORF sequences indicate that the CAL serogroup bunyaviruses can be divided into three monophyletic lineages corresponding to three of the complexes previously derived by serological classification. The truncated version of the NSs protein, which is found in five CAL serogroup bunyaviruses, appears to have arisen twice during virus evolution.

Identification of a coat protein binding site on southern bean mosaic virus RNA.

The cowpea strain of Southern bean mosaic virus (SBMV-C), a T = 3 icosahedral RNA virus, was dissociated to yield a ribonucleoprotein complex (RNPC) composed of the viral RNA and coat protein subunits. To determine if the coat protein subunits were bound to a specific site on the viral RNA, the RNPC was treated with ribonuclease, and the remaining coat protein--RNA complexes were recovered by filter binding. A single species of RNA was isolated by this procedure and further characterized by sequencing. The RNA was mapped to nucleotides 1410-1436 of the SBMV-C genome. This region of the viral RNA was predicted to fold into a hairpin with a 4-base loop and a duplex stem of 24 nucleotides. The stability and specificity of the coat protein-RNA complex isolated from dissociated virus suggest a possible role for this interaction in the selective encapsidation of the viral RNA.

Intracranial extension of a nasal dermoid sinus cyst in a 56-year-old man.

BACKGROUND: Nasal dermoid sinus cysts are rare congenital lesions that result from aberrant embryonal development. Cases of nasal dermoids associated with intracranial extension have been reported in the past, but have almost always occurred in children. METHODS: One case of a nasal dermoid cyst with intracranial involvement in a 56-year-old man is reported herein. RESULTS: A 56-year-old man presented with a draining sinus in the right medial canthal area and a nasal pit. Imaging studies suggested a nasal dermoid cyst with intracranial extension. He subsequently underwent a craniofacial removal of the lesion with a satisfactory postoperative course. CONCLUSION: That nasal dermoid cysts can remain dormant until the age of 56 suggests that the incidence of this lesion with intracranial extension may be higher than previously reported. A one-stage combined otolaryngologic/neurosurgical procedure is advocated for those lesions that are extra- and intracranial.

Use of the mental health on-call system on a university campus.

Over the course of 1 calendar year, clinicians at a university mental health service collected data on every clinical case in which students presented after normal business hours or on weekends for urgent mental health consultation. During the year, 50 such incidents were recorded, which translated to a rate of 6.6 on-call events per year per 1,000 students. Students were primarily self-referred or referred by the student health center or residence life staff. Suicidal concerns, panic/anxiety, and depressive states were the three most common presenting complaints. Average clinician time per case was 1 1/2 hours, with sexual assault cases taking the most time per case, followed by substance abuse and suicidality. Follow-up outpatient counseling was employed in 76% of the cases. The results highlight the importance of on-call mental health services on college campuses.

Incidence of cancer among patients with rheumatoid arthritis.

BACKGROUND: To evaluate hypotheses about the relationship between immune alterations and cancer, several investigators have determined cancer incidence in groups of patients with rheumatoid arthritis (RA), a chronic autoimmune disease. The primary finding has been an increased risk of hematopoietic cancers. PURPOSE: In this study, we have attempted to refine estimates of the association between RA and subsequent development of specific cancers. METHODS: We investigated site-specific cancer risk associated with RA in a population-based cohort study of 11683 Swedish men and women with a hospital (inpatient) diagnosis of RA. These case patients were identified from 1965 to 1983 and had follow-up through 1984 by computer linkage of the Swedish Hospital Inpatient Register to the National Swedish Cancer Registry (840 case patients with cancer) and the Swedish Registry of Causes of Death. Cancer risk was estimated by standardized incidence ratios (SIRs) for specific cancers. RESULTS: For men and women overall, there were decreased risks for cancers of the colon (SIR = 0.63; 95% confidence interval [CI] = 0.5-0.9), rectum (SIR = 0.72; 95% CI = 0.5-1.1), and stomach (SIR = 0.63; 95% CI = 0.5-0.9) and an increased risk for lymphomas (SIR = 1.98; 95% CI = 1.5-2.6). CONCLUSIONS: The reduced risk for colorectal cancer in patients with RA is consistent with previous studies of RA patients and with reports which state that use of nonsteroidal anti-inflammatory drugs may protect against the development of large bowel cancers. The excess of lymphomas also confirms a number of earlier investigations of RA patients.

Characterization of an internal element in turnip crinkle virus RNA involved in both coat protein binding and replication.

The major coat-protein-binding element of turnip crinkle virus RNA was previously mapped in the region of the UAG termination codon in the viral polymerase gene. This region encompasses two of the high-affinity coat-protein-binding sites (Fa and Ff) that we suggested were physically associated in a stem-loop in a ribonucleoprotein complex involved in assembly initiation (Wei, Heaton, Morris, and Harrison, J. Mol. Biol. 214, 85-95, 1990). We have also demonstrated that this RNA element was capable of specific coat protein binding in vitro (Wei and Morris, J. Mol. Biol. 222, 437-443, 1991). We now provide physical evidence, by in vitro chemical and enzymatic probing of the viral RNA, that support the suggestion that the two coat-protein-binding sites base pair to form a stem structure (A/F stem) surrounding the UAG terminator in wild-type RNA. We have shown here that a mutant with seven conservative nucleotide substitutions in Fa does not accumulate to detectable levels in plants or protoplasts and that the A/F stem structure is drastically altered in this mutant. We suggest that the primary effect of this mutation is on replication rather than on a reduction in RNA stability resulting from a defect in encapsidation of the virion RNA because previous results have shown that encapsidation-deficient mutants have little or no effect on viral RNA replication (Hacker, Petty, Wei, and Morris, Virology 186, 1-8, 1992). The analysis of the A/F stem was extended by construction and characterization of a series of mutants and revertants that displayed variable levels of replication deficiency but minimal concomitant defect in encapsidation efficiency. The extent of the replication defect correlated with the predicted destabilization of the A/F stem structure. We conclude from these results that this RNA element is involved in viral replication, and we tentatively suggest that the A/F stem structure may be functionally involved in the readthrough translation of the viral polymerase.

Alcoholism and liver cirrhosis in the etiology of primary liver cancer.

The aim of this study was to determine the risk of developing primary liver cancer in patients with a diagnosis of alcoholism, liver cirrhosis, or both. Three population-based, mutually exclusive cohorts were defined on the basis of hospital discharge diagnosis between 1965 and 1983. Complete follow-up through 1984--excluding the first year of follow-up--showed that among 8,517 patients with a diagnosis of alcoholism, 13 cancers occurred, vs. 4.2 expected (standardized incidence ratio (SIR) = 3.1; 95% confidence interval (CI) = 1.6 to 5.3); among 3,589 patients with liver cirrhosis, 59 cancers occurred, vs. 1.7 expected (SIR = 35.1; 95% CI = 26.7 to 45.3), and among 836 patients with both diagnoses, 11 cancers occurred, vs. 0.3 expected (SIR = 34.3; 95% CI = 17.1 to 61.3). Thus, alcoholism alone entailed a moderately increased risk and alcoholism with liver cirrhosis did not increase the high relative risk for liver cancer more than cirrhosis alone. We conclude that alcohol intake may be a liver carcinogen only by being causally involved in the development of cirrhosis; and further, that the risk of developing liver cancer following cirrhosis in this population is similar to or higher than that after chronic hepatitis-B-virus infection in other Western countries.

Single amino acid change in the helicase domain of the putative RNA replicase of turnip crinkle virus alters symptom intensification by virulent satellites.

The virulent satellite [satellite C (sat C)] of turnip crinkle virus (TCV) is a small pathogenic RNA that intensifies symptoms in TCV-infected turnip plants (Brassica campestris). The virulence of sat C is determined by properties of the satellite itself and is influenced by the helper virus. Symptoms produced in infections with sat C differ in severity depending on the helper virus. The TCV-JI helper virus produces more severe symptoms than the TCV-B helper virus when inoculated with sat C. To find determinants in the TCV helper virus genome that affect satellite virulence, the TCV-JI genome was cloned and the sequence compared to the TCV-B genome. The genomes were found to differ by only five base changes, and only one of the base changes, at nucleotide position 1025, produced an amino acid change, an aspartic acid----glycine in the putative viral replicase. A chimeric TCV genome (TCV-B/JI) containing four of the five base changes (including the base change at position 1025) and a mutant TCV-B genome (TCV-B1025G) containing a single base substitution at position 1025 converted the TCV-B genome into a form that produces severe symptoms with sat C. The base change a position 1025 is located in the helicase of the putative viral replicase, and symptom intensification appears to result from differences in the rate of replication of the satellite supported by the two helper viruses.