Fine needle aspiration cytology of the thyroid.

This article is an up-to-date review of thyroid fine needle aspiration. Special emphasis is placed on pitfalls and differential diagnoses of common and uncommon thyroid lesions.

T-cell-rich B-cell lymphoma and Epstein-Barr virus infection of the uterus in a postmenopausal patient with an intrauterine contraceptive device in place for over 20 years.

Although secondary involvement of the female genital tract occurs in up to 40% of cases of disseminated lymphomas, lymphomas presenting with primary female genital tract symptomatology are very unusual. We report a case of T-cell-rich B-cell lymphoma (TCRBCL) arising in the uterine corpus of a 57-year-old female who carried an intrauterine contraceptive device (IUD) for over 20 years. Malignant lymphoid cells expressed the Epstein-Barr virus (EBV) late membrane protein (LMP), a feature described in TCRBCL but not previously reported in primary uterine lymphomas. To our knowledge, this is the first reported case of a TCRBCL of the uterus.

Reversion by deletion of transforming oncogene following interferon-beta and retinoic acid treatment.

We have previously shown that prolonged interferon-beta (IFN-beta) treatment of RS485 cells (NIH3T3 cells transformed with multiple copies of an LTR-cHa-ras oncogene) resulted in the phenotypic reversion of 1%-5% of the culture, depending on the conditions used. This reversion persisted after IFN-beta was discontinued, although the revertants retained the LTR-cHa-ras and continued to express ras mRNA and p21. Clones were prepared of such persistent revertant cell lines (PRs). Expression of lysyl oxidase (LOX), which appears to act as a suppressor of ras transformation, was downregulated in RS485 and upregulated in the PRs. When retinoic acid (RA) was combined with IFN-beta treatment of the RS485 cultures, a different mechanism of reversion predominated. Following 60 days of treatment with 20 IU/ml of IFN-beta and 10 microM RA, all of the multiple (3-5) copies of the transforming LTR-c-Ha-ras originally present in RS485 cells were deleted from the genome in 72% of 54 revertant cell lines isolated. As in the case of revertants observed after treatment with IFN-beta alone, LOX mRNA expression was upregulated in all of the revertants that resulted from the treatment with IFN plus RA. The level of LOX mRNA expression acts, therefore, as an indicator of transformation in this system.

Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls.

A number of reports have indicated that RNA recovered from paraffin-embedded tissue can be used as a substrate in the polymerase chain reaction (PCR). Although it is established that RNA in paraffin-embedded tissue undergoes significant degradation, the specific contributions of different fixatives and fixation times to this degradation are not known. Mouse splenic tissue was harvested and fixed immediately for 2, 8, or 24 h in either formalin, Omnifix II, or Carnoy's fixative and then processed and embedded in paraffin. RNA was extracted from deparaffinized cubes of tissue using an adaptation of the technique described by Chomczynski and Sacchi. RNA was reverse transcribed using a random hexamer primed reaction. PCR amplification for cDNAs of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (HPRT) mRNAs was then performed. Although GAPDH amplification is used routinely on fresh and frozen tissues, we show that the presence of DNA contamination in the RNA preparations limits its usefulness in paraffin-embedded tissue. Amplifiable HPRT mRNA sequences were detected in nine of 12 samples fixed in Omnifix II, in four of 12 samples fixed in Carnoy's fixative, and in none of 12 formalin-fixed samples. Because of primer selection to preclude amplification of genomic HPRT, DNA contamination is not an issue when HPRT is amplified. Thus, HPRT represents the control system of choice for the evaluation of RNA in PET. The techniques described provide a rapid, uniform, and reproducible method of obtaining RNA from PET for molecular analysis, but they indicate limited utility for retrospective analysis of archival tissues.

Sequencing, targeted mutagenesis and expression of a recA gene required for the extreme radioresistance of Deinococcus radiodurans.

Deinococcus radiodurans and other members of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. A DNA damage-sensitive and natural transformation-deficient strain generated by chemical mutagenesis (strain rec30) was found to be defective in a gene that has extended homology with recA of Escherichia coli. Upon transformation with a chromosomal DNA fragment that contained this deinococcal recA gene from wild-type (wt) D. radiodurans both DNA damage resistance and full transformation competence were restored in the rec30 mutant. Targeted insertional mutagenesis of the deinococcal recA gene was used to construct a mutant isogenic with the wt. The insertional mutant was phenotypically indistinguishable from strain rec30, indicating that the recA defect alone was responsible for observed phenotypic alterations. For example, in the case of ionizing radiation, the D37 of the wt was about 1.75 Mrad, while the D37 of rec30 and the insertional mutant were both 25 krad, a 70-fold decrease. Evidence is presented that expression of the deinococcal recA gene in E. coli is lethal, suggesting that the mode of interaction of the deinococcal RecA protein with nucleic acids or other cellular proteins differs at least in part from RecA of E. coli.

Restoration of the DNA damage resistance of Deinococcus radiodurans DNA polymerase mutants by Escherichia coli DNA polymerase I and Klenow fragment.

Deinococcus radiodurans and other species of this genus share extreme resistance to ionizing radiation and many other agents that damage DNA. D. radiodurans mutant strains defective in a deinococcal DNA polymerase that is homologous with E. coli DNA polymerase I are highly sensitive to DNA damage. In the current work we have inquired whether E. coli DNA Pol I can substitute for D. radiodurans Pol in partially or fully restoring to pol- D. radiodurans mutants the extreme DNA damage-resistance typical of this organism. The E. coli polA gene or a 5'-truncated polA gene that encodes the Klenow fragment were introduced and expressed in two different D. radiodurans pol- mutants: Strain 303, which is a chemically mutagenized derivative, and strain 6R1A, which is isogenic with wild-type D. radiodurans except for an insertional mutation within the pol gene. Expression of E. coli polA in both of these mutants fully restored wild-type resistance to ionizing- and UV254-radiation and mitomycin-C exposure. Expression of the Klenow fragment-encoding gene restored wild-type resistance to D. radiodurans strain 303, but only partial resistance to strain 6R1A. The observation that E. coli DNA Pol I is as effective as D. radiodurans Pol in restoring damage resistance, indicates that D. radiodurans DNA Pol per se does not have special properties that are essential or prerequisite for expression of the extreme resistance of D. radiodurans.

Identification, sequencing, and targeted mutagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans.

Deinococcus radiodurans and other species of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. Two different DNA damage-sensitive strains generated by chemical mutagenesis were found to be defective in a gene that has extended DNA and protein sequence homology with polA of Escherichia coli. Both mutant strains lacked DNA polymerase, as measured in activity gels. Transformation of this gene from wild-type D. radiodurans restored to the mutants both polymerase activity and DNA damage resistance. A technique for targeted insertional mutagenesis in D. radiodurans is presented. This technique was employed to construct a pol mutant isogenic with the wild type (the first example of targeted mutagenesis in this eubacterial family). This insertional mutant lacked DNA polymerase activity and was even more sensitive to DNA damage than the mutants derived by chemical mutagenesis. In the case of ionizing radiation, the survival of the wild type after receiving 1 Mrad was 100% while survival of the insertional mutant extrapolated to 10(-24). These results demonstrate that the gene described here encodes a DNA polymerase and that defects in this pol gene cause a dramatic loss of resistance of D. radiodurans to DNA damage.

Increased resistance to ionizing and ultraviolet radiation in Escherichia coli JM83 is associated with a chromosomal rearrangement.

Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).

Binding of erythroid and non-erythroid nuclear proteins to the silencer of the human epsilon-globin-encoding gene.

To clarify the molecular mechanisms involved in the developmental control of hemoglobin-encoding genes we have been studying the expression of these genes in human cells in continuous culture. We have previously reported the presence of a transcriptional control element with the properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin-encoding gene [Cao et al., Proc. Natl. Acad. Sci. USA 86 (1989) 5306-5309]. We also showed that this silencer has stronger inhibitory activity in HeLa cells, as compared to K562 human erythroleukemia cells. Using deletion mutants and cis-cloned synthetic oligodeoxyribonucleotides in transient expression assays, nucleotide sequences responsible for this effect have now been further delimited to 44 bp located from -294 to -251 bp. Gel electrophoresis mobility shift assays and DNaseI footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. Two binding proteins are detected in K562 nuclear extracts, while only one is found in extracts from HeLa cells. Possible mechanisms by which these proteins may regulate transcription of the epsilon-globin-encoding gene in erythroid and non-erythroid cells are discussed.

Heterozygosity and instability of amplified chromosomal insertions in the radioresistant bacterium Deinococcus radiodurans.

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. The circles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strains that are defective in natural transformation and sensitive to DNA-damaging agents were further characterized. Both strains were defective in duplication insertion. While on strain was normal for plasmid transformation, the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may require recombinational functions.

Evidence against enhancement of the radioresistance of Escherichia coli by cloned Deinococcus radiodurans DNA.

Deinococcus radiodurans genomic DNA, introduced to Escherichia coli in cloning vectors, has been reported to produce radioresistant E. coli that can be selected by gamma irradiation. In this report prior results are reassessed experimentally, and additional studies are presented. Results to date suggest that the acquired radioresistance of E. coli selected by gamma irradiation does not stem from expression of stable plasmid-encoded D. radiodurans sequences, and that acquired radioresistance is not readily transmitted to naive (unirradiated) E. coli by transformation of plasmid recovered from the radioresistant isolates. Several interpretations are discussed.

Partial complementation of the UV sensitivity of Deinococcus radiodurans excision repair mutants by the cloned denV gene of bacteriophage T4.

Deinococcus radiodurans has 2 endonucleases that incise UV-irradiated DNA. UV endonuclease-alpha and UV endonuclease-beta, that are believed to functionally overlap. Both endonucleases must be mutationally inactivated to yield an incisionless, markedly UV-sensitive phenotype. denV, the bacteriophage T4 gene encoding pyrimidine dimer-DNA glycosylase (PD-glycosylase), was introduced and expressed via duplication insertion in D. radiodurans wild-type, and single and double UV endonuclease mutants. The strain deficient in UV endonuclease-alpha has wild-type UV resistance, and the expression of PD-glycosylase exerted no survival effect on this strain or wild-type. Expression of denV increased survival of both the markedly UV-sensitive double mutant and the moderately UV-sensitive strain deficient only in UV endonuclease-beta. In endonuclease-beta-deficient cells phenotypic complementation by denV was almost complete in restoring UV resistance to wild-type levels. These results suggest that UV endonuclease-alpha (which is present in the endonuclease-beta-deficient cells) does not recognize one or more types of cyclobutane dimer incised by the PD-glycosylase or UV endonuclease-beta.

A highly conserved repeated chromosomal sequence in the radioresistant bacterium Deinococcus radiodurans SARK.

A DNA fragment containing a portion of a DNA damage-inducible gene from Deinococcus radiodurans SARK hybridized to numerous fragments of SARK genomic DNA because of a highly conserved repetitive chromosomal element. The element is of variable length, ranging from 150 to 192 bp, depending on the absence or presence of one or two 21-bp sequences located internally. A putative translational start site of the damage-inducible gene is within the reiterated element. The element contains dyad symmetries that suggest modes of transcriptional and/or translational control.

Trans-activation of human globin genes by HTLV-I tax1.

We studied the effects of a known retroviral trans-activating factor, HTLV-I tax1, on transcription of human globin genes. Transfection of HeLa cells by the cloned tax1 gene stimulated activity of both the beta- and epsilon-globin promoters approximately 20-fold, as measured by chloramphenicol acetyl transferase (CAT) assays. Studies of promoter 5'-deletion mutants revealed that the trans-activation response required only 185 base pairs (bp) of beta-globin 5'-flanking sequence or 177 bp of epsilon-globin 5' flanking sequence. These promoter regions contain either two (for beta) or three (for epsilon) copies of the pentanucleotide sequence CTGAC, which is characteristic of previously described tax1-responsive promoters. We also stably transfected tax1 into the erythroid cell line K562. Transfectants expressing tax1 showed increased transcription of epsilon-, gamma-, zeta-, and alpha-globins. This indicates that tax1 can stimulate transcription of globin genes in their native chromosomal location. This was confirmed by measurements of increases in intracellular hemoglobin as determined by an increased percentage of cells staining with benzidine and by spectrophotometric measurements of hemoglobin. The observed trans-activation of globin genes by tax1 may provide insight into normal regulation of globin genes by clarifying cis regulatory sequences. Furthermore, it suggests that the trans-acting effects of tax1 on heterologous genes are more widespread than was previously appreciated.

Identification of a transcriptional silencer in the 5'-flanking region of the human epsilon-globin gene.

We have studied the 5'-flanking sequences required for the transcriptional regulation of human epsilon-globin gene expression. A series of deletion mutants of the human epsilon-globin gene 5'-flanking sequences were constructed and linked to the bacterial chloramphenicol acetyltransferase gene. Expression of these constructs was tested in HeLa cells and the human erythroleukemia K-562 cells. By measuring chloramphenicol acetyltransferase activities and mRNA levels we found that the sequence between -177 and -392 base pairs (bp) relative to the mRNA initiation site exerts a negative effect on epsilon-globin promoter activity. This effect is more pronounced in HeLa cells compared with K-562 cells. To further characterize the negative control region we cloned the DNA sequence between -177 and -392 bp either 5' or 3' of the epsilon-globin promoter and in either orientation. Our data indicate that this negative control region inhibits the epsilon-globin promoter activity in a position- and orientation-independent manner, thus suggesting that it is a silencer. In addition, the silencer also inhibits the expression from the Herpesvirus thymidine kinase promoter. Sequence comparison reveals that there are three short regions within the silencer that share extensive homology with those found in other negative control DNA elements. Our results therefore indicate that an upstream silencer element is present in the epsilon-globin gene and that it may play an important role in the control of epsilon-globin gene expression during development.