Nonrandom gene organization: structural arrangements of specific pre-mRNA transcription and splicing with SC-35 domains.

This work demonstrates a highly nonrandom distribution of specific genes relative to nuclear domains enriched in splicing factors and poly(A)+ RNA, and provides evidence for the direct involvement of these in pre-mRNA metabolism. As investigated in hundreds of diploid fibroblasts, human collagen I alpha 1 and beta-actin DNA/RNA showed a very high degree of spatial association with SC-35 domains, whereas three nontranscribed genes, myosin heavy chain, neurotensin, and albumin, showed no such preferential association. Collagen I alpha 1 RNA accumulates within the more central region of the domain, whereas beta-actin RNA localizes at the periphery. A novel approach revealed that collagen RNA tracks are polarized, with the entire gene at one end, on the edge of the domain, and the RNA extending into the domain. Intron 26 is spliced within the RNA track at the domain periphery. Transcriptional inhibition studies show both the structure of the domain and the gene's relationship to it are not dependent upon the continued presence of accumulated collagen RNA, and that domains remaining after inhibition are not just storage sites. Results support a model reconciling light and electron microscopic observations which proposes that transcription of some specific genes occurs at the border of domains, which may also function in the assembly or distribution of RNA metabolic components. In contrast to the apparently random dispersal of total undefined hnRNA synthesis through interdomain space, transcription and splicing for some genes occurs preferentially at specific sites, and a high degree of individual pre-mRNA metabolism is compartmentalized with discrete SC-35 domains.

XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure.

The XIST gene is implicated in X chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2- and 3-D space; hence, the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its polyadenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of nonchromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging.

Interphase and metaphase resolution of different distances within the human dystrophin gene.

Fluorescence in situ hybridization makes possible direct visualization of single sequences not only on chromosomes, but within decondensed interphase nuclei, providing a potentially powerful approach for high-resolution (1 Mb and below) gene mapping and the analysis of nuclear organization. Interphase mapping was able to extend the ability to resolve and order sequences up to two orders of magnitude beyond localization on banded or unbanded chromosomes. Sequences within the human dystrophin gene separated by less than 100 kb to 1 Mb were visually resolved at interphase by means of standard microscopy. In contrast, distances in the 1-Mb range could not be ordered on the metaphase chromosome length. Analysis of sequences 100 kb to 1 Mb apart indicates a strong correlation between interphase distance and linear DNA distance, which could facilitate a variety of gene-mapping efforts. Results estimate chromatin condensation up to 1 Mb and indicate a comparable condensation for different cell types prepared by different techniques.

A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus.

A quantitative three-dimensional analysis of nuclear components involved in precursor messenger RNA metabolism was performed with a combination of fluorescence hybridization, immunofluorescence, and digital imaging microscopy. Polyadenylate [poly(A)] RNA-rich transcript domains were discrete, internal nuclear regions that formed a ventrally positioned horizontal array in monolayer cells. A dimmer, sometimes strand-like, poly(A) RNA signal was dispersed throughout the nucleoplasm. Spliceosome assembly factor SC-35 localized within the center of individual domains. These data support a nuclear model in which there is a specific topological arrangement of noncontiguous centers involved in precursor messenger RNA metabolism, from which RNA transport toward the nuclear envelope radiates.

Prediction of rho-independent transcriptional terminators in Escherichia coli.

A new algorithm called RNAMotif containing RNA structure and sequence constraints and a thermodynamic scoring system was used to search for intrinsic rho-independent terminators in the Escherichia coli K-12 genome. We identified all 135 reported terminators and 940 putative terminator sequences beginning no more than 60 nt away from the 3'-end of the annotated transcription units (TU). Putative and reported terminators with the scores above our chosen threshold were found for 37 of the 53 non-coding RNA TU and for almost 50% of the 2592 annotated protein-encoding TU, which correlates well with the number of TU expected to contain rho-independent terminators. We also identified 439 terminators that could function in a bi-directional fashion, servicing one gene on the positive strand and a different gene on the negative strand. Approximately 700 additional termination signals in non-coding regions (NCR) far away from the nearest annotated gene were predicted. This number correlates well with the excess number of predicted 'orphan' promoters in the NCR, and these promoters and terminators may be associated with as yet unidentified TU. The significant number of high scoring hits that occurred within the reading frame of annotated genes suggests that either an additional component of rho-independent terminators exists or that a suppressive mechanism to prevent unwanted termination remains to be discovered.

Structure and partial genomic sequence of the human E2F1 gene.

The E2F family of transcription factors appears to play a critical role in the transcription of certain genes required for cell cycle progression. E2F1, the first cloned member of this family, is regulated during the cell cycle at the mRNA level by changes in transcription of the E2F1 gene and at the protein level by complex formation with proteins such as the retinoblastoma gene product (pRB), cyclin A and DP1. E2F1 can override a pRB-induced G1/S block and can behave as an oncogene in certain cells. E2F1 was cloned and was found to contain seven exons. The dinucleotides at the 5' and 3' splice sites of intron 4 do not agree with consensus splice site sequences. Fluorescence in situ hybridization localized E2F1 to chromosome 20q11. Knowledge of the organization of E2F1 may facilitate identification of additional E2F family members, as well as detection of E2F1 abnormalities in human tumors.

Sex differences in bipolar affective disorder: neuroleptic dosage variance.

Bipolar affective disorder in men and women often differs in prevalence, age of onset, phenomenology, and longitudinal course. A study of 112 bipolar patients, comprising 72 women and 40 men who were discharged from an acute inpatient setting on antipsychotic drugs, is reported. Higher mean discharge neuroleptic doses were prescribed to men below the age of 40 and to women above the age of 40. The clinical implications of higher dosing patterns are discussed.

Seasons and bipolar disorder.

The records of 377 bipolar disorder patients who were consecutively admitted to a general inpatient psychiatric unit in mid-Michigan over a 6-year period were examined. The seasonal variation of hospitalization, total sleep time, thyroid stimulating hormone, creatinine levels, lithium dosage and serum levels, aggressive behavior, and treatment outcome were analyzed. Among men, the admission rate peaked in the springtime. Women demonstrated a bimodal season distribution, with peak admission rates in spring and fall. Aggressive behavior in both men and women peaked in the spring (z = 2.50, p < 0.05). Men maintained on lithium achieved higher serum lithium levels during the summer months. These findings parallel previous reports regarding the influence of seasons upon bipolar disorder. The therapeutic implications related to seasonality and mania are discussed.

Chromosomal assignment of human nuclear envelope protein genes LMNA, LMNB1, and LBR by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization to establish precise chromosomal localizations for three human genes encoding four different nuclear envelope proteins. Lamin A/C (LMN1, HGMW-approved symbol LMNA) mapped to 1q21.2-q21.3, with a most probable gene assignment to 1q21.3; lamin B receptor (LBR) was localized to 1q42.1; and lamin B1 (LMNB1) was mapped to the interface of bands 5q23.3-q31.1. Assignments were determined by direct placement of signals relative to high-resolution DAPI or G-bands. Comparison of these results of band positions predicted from fractional length measurements to signal placement indicated that more accurate predictions are made using Francke idiograms and that measurement strategy avoids variance due to polymorphic chromosome segments.

Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization.

The enormous potential of in situ hybridization derives from the unique ability of this approach to directly couple cytological and molecular information. In recent years, there has been a surge of success in powerful new applications, resulting from methodologic advances that bring the practical capabilities of this technology closer to its theoretical potential. A major advance has been improvements that enable, with a high degree of reproducibility and efficiency, precise visualization of single sequences within individual metaphase and interphase cells. This has implications for gene mapping, the analysis of nuclear organization, clinical cytogenetics, virology, and studies of gene expression. This article discusses the current state of the art of fluorescence in situ hybridization, with emphasis on applications to human genetics, but including brief discussions on studies of nuclear DNA and RNA organization, and on applications to clinical genetics and virology. Although a review of all of the literature in this field is not possible here, many of the major contributions are summarized along with recent work from our laboratory.

When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates.

In this study we used Gardella gel analysis of intact DNA, Southern blotting of digested DNA, and fluorescence in situ hybridization to provide complementary and unequivocal information on the state of the Epstein-Barr virus (EBV) genome in persistently infected cells. The fluorescence in situ hybridization technique allowed us to directly visualize both integrated and episomal EBV DNA at the single-cell level. We show here that circularization of the EBV genome is rarely detected upon infecting activated normal B cells. The virus can persist upon infection of a different proliferating B-cell target, EBV-negative Burkitt's lymphoma tumor cell lines. Analysis of 16 such lines reveal again, that the virus infrequently persists as covalently closed episomes; rather, the virus preferentially persists by integrating into the host DNA (10 of 16 clones). The integrated virus is linear and usually intact, although 3 of 10 isolates have deletions from the left-hand end including the latent origin of replication. At the level of our analysis, no obvious relationship was seen between the integration sites. These studies provide, for the first time, a reproducible in vitro model system to study integration by EBV.

Homologous ribosomal protein genes on the human X and Y chromosomes: escape from X inactivation and possible implications for Turner syndrome.

We have isolated two genes on the human sex chromosomes, one on the Y and one on the X, that appear to encode isoforms of ribosomal protein S4. These predicted RPS4Y and RPS4X proteins differ at 19 of 263 amino acids. Both genes are widely transcribed in human tissues, suggesting that the ribosomes of human males and females are structurally distinct. Transcription analysis revealed that, unlike most genes on the X chromosome, RPS4X is not dosage compensated. RPS4X maps to the long arm of the X chromosome (Xq), where no other genes are known to escape X inactivation. Curiously, RPS4X maps near the site from which the X-inactivating signal is thought to emanate. On the Y chromosome, RPS4Y maps to a 90 kb segment that has been implicated in Turner syndrome. We consider the possible role of RPS4 haploinsufficiency in the etiology of the Turner phenotype.