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PubMed1991

Integration of woodchuck hepatitis virus (WHV) DNA at two chromosomal sites (Vk and gag-like) in a hepatocellular carcinoma.

Integration of woodchuck hepatitis virus (WHV) DNA into the liver DNA of a woodchuck infected by the virus was investigated. Clonal viral integration was not detected three months before the appearance of four hepatocellular carcinomas (HCC). Integration of the viral DNA was detected in all four HCCs, of which one was chosen to determine the structure of the viral integration completely in a single tumor. The integration occurred in two sites. One part contains the viral DNA from the middle of the gene encoding surface antigen to two-thirds of the way through the gene encoding X protein (X) with no structural changes. The coding frame of the truncated X gene continues into the chromosomal sequence to make a possible fusion protein. The integration seems to have occurred by recombination within two direct repeats of the viral genome in one junction and by homologous recombination between viral DNA and chromosomal DNA in the other junction. The viral DNA is integrated into a spacer of the immunoglobulin L-chain Vk (IgVk) region without any chromosomal rearrangement accompanying the integration. The viral DNA at the second site has a complex structural rearrangement: part of the preS gene is duplicated and attached to the terminus of the gene encoding core antigen in a head-to-tail fashion, followed by three small fragments derived from other parts of the viral DNA. The integrated preS gene has its own 5' regulatory element and a coding frame consisting of the truncated preS gene, the other parts of viral DNA and the chromosomal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2006

Relation of social integration to inflammatory marker concentrations in men and women 70 to 79 years.

The lack of social integration has predicted coronary heart disease morbidity and mortality in prospective observational studies; however, the biologic mechanisms by which this may occur are not well understood. The objective of this study was to determine whether social integration is associated with inflammatory risk factors for coronary heart disease, specifically C-reactive protein (CRP) and interleukin-6. The study participants (aged 70 to 79 years; 380 men and 425 women) were from the MacArthur Successful Aging Study, a longitudinal study of 3 community-based cohorts in the United States of older adults with relatively high physical and cognitive functioning at baseline (1988 to 1989). The plasma concentrations of interleukin-6 and CRP were assessed using a high-sensitivity enzyme-linked immunosorbent assay. Cross-sectional logistic regression analyses were performed. Multivariate adjusted analyses indicated that social integration was significantly inversely associated with CRP concentration in men after adjusting for age, race/ethnicity, smoking, alcohol consumption, physical activity, body mass index, cardiovascular disease, other major or chronic conditions, physical functioning, socioeconomic status, and depression (odds ratio 2.23, 95% confidence interval 1.05 to 4.76, for elevated CRP [>3.19 mg/L] in the least socially integrated quartile vs the most socially integrated quartile). No significant associations were found between social integration and interleukin-6 in men or either inflammatory marker in women. In conclusion, social integration was negatively associated with the plasma CRP concentration in men. These findings suggest a potential biologic mechanism for the observed associations between social integration and coronary heart disease in prospective studies. Differences may exist between women and men in the biologic pathways associated with social integration.

Age Factors↗
PubMed2004

Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors.

Uncontrolled insertion of gene transfer vectors into the human genome is raising significant safety concerns for their clinical use. The wild-type adeno-associated virus (AAV) can insert its genome at a specific site in human chromosome 19 (AAVS1) through the activity of a specific replicase/integrase protein (Rep) binding both the AAVS1 and the viral inverted terminal repeats (ITRs). AAV-derived vectors, however, do not carry the rep gene and cannot maintain site-specific integration properties. We describe a novel hybrid vector carrying an integration cassette flanked by AAV ITRs and a tightly regulated, drug-inducible Rep expression cassette in the framework of a high-capacity, helper-dependent adenoviral (Ad) vector. Rep-dependent integration of ITR-flanked cassettes of intact size and function was obtained in human primary cells and cell lines in the absence of selection. The majority of integrations were site specific and occurred within a 1000-bp region of the AAVS1. Genome-wide sequencing of integration junctions indicates that nonspecific integrations occurred predominantly in intergenic regions. Site-specific integration was obtained also in vivo, in an AAVS1 transgenic mouse model: upon a single tail vein administration of a nontoxic dose of Ad/AAV vectors, AAVS1-specific integrations were detected and sequenced in DNA obtained from the liver of all animals in which Rep expression was induced by drug treatment. Nonrandom integration of double-stranded DNA can therefore be obtained ex vivo and in vivoby the use of hybrid Ad/AAV vectors, in the absence of toxicity and with efficiency compatible with gene therapy applications.

Adenoviridae↗
PubMed1998

An integrated approach to the quantification of aortic regurgitation by Doppler echocardiography.

BACKGROUND: Although different Doppler methods have been proposed for the quantification of aortic regurgitation, no study has prospectively compared these methods with each other and their correlation with angiography. The aim of this study was to prospectively analyze the usefulness of different Doppler echocardiography parameters by testing all such parameters in each patient. METHODS: Fifty-one patients with aortic regurgitation underwent 2-dimensional and Doppler echocardiographic studies and catheterization. The following Doppler indexes were analyzed and compared with aortography. Color Doppler: (1) jet color height/left ventricular outflow tract height in parasternal long-axis view, and (2) jet color area/left ventricular outflow tract area in short-axis view. Continuous Doppler: (3) regurgitant flow pressure half-time, (4) regurgitant flow time velocity integral (in centimeters), and (5) regurgitant flow time velocity integral (in centimeters)/diastolic period (in milliseconds). Pulsed Doppler in thoracic and abdominal aorta: (6) time velocity integral of diastolic reverse flow (in centimeters), (7) time velocity integral of systolic anterograde flow/integral of diastolic reverse flow, (8) (time velocity integral of diastolic reverse flow/diastolic period) x 100, and (9) diastolic reverse flow duration/diastolic period (as a percentage). We compared these parameters with severity of regurgitation measured by angiography and classified as mild, moderate, or severe. RESULTS: The most useful parameters were (1) jet color height/left ventricular outflow tract height (correctly classified 42 of 49 patients), (2) (time velocity integral of diastolic reverse flow/diastolic period) x 100 in the thoracic aorta (correctly classified 41 of 46 patients), and (3) (time velocity integral of diastolic reverse flow/diastolic period) x 100 in the abdominal aorta (correctly classified 42 of 49 patients). Sequential integration of these 3 parameters correctly classified 96% of patients (44 of 46 patients) and was achieved in 90% of cases. CONCLUSION: An integrated combination of several Doppler parameters can quickly and accurately classify the degree of aortic regurgitation as determined by angiography.

Aged↗
PubMed1986

Integration of HBV-DNA may not be a prerequisite for the maintenance of the state of malignant transformation. An analysis of 110 liver biopsies.

Hundred and ten liver biopsy specimens from various parts of the world were examined for episomal and integrated HBV-DNA sequences. In 54 patients with HBsAg chronic liver disease episomal HBV-DNA was found in 83% of HBeAg-positive patients, compared to only 22% of patients with anti-HBe. Furthermore episomal HBV-DNA in the latter predominated among the Asians. Integrated HBV-DNA was found only in 5.5% of HBeAg-positive patients but in 16.5% of patients with anti-HBe. In 28 HBsAg-positive patients with hepatoma, episomal HBV-DNA was found in 50% of HBeAg-positive patients but in only 11% of anti-HBe patients. Conversely integrated sequences were less common (25%) in HBe-Ag-positive patients than in anti-HBe patients (50%), giving an overall incidence of integration in this group of 45%. No episomal, and only one case of integrated sequences of HBV-DNA, could be detected among 10 patients with HBsAg-negative hepatoma. In addition neither episomal nor integrated HBV-DNA could be detected in 18 patients with non-HBV-related liver disease. Our data suggests that stable integration of HBV-DNA into the host's genome is not necessarily a prerequisite for the maintenance of the state of malignant transformation but may be necessary for its initiation. Alternatively, the detection of integrated HBV-DNA may represent a 'snap shot' of a random integration event amplified by clonal expansion promoted by other factors.

Base Sequence↗
PubMedDate not supplied

Quantitative myocardial ultrasonic integrated backscatter measurements during contrast injections.

We and others have shown that normal myocardium exhibits 4 to 5 dB diastolic-to-systolic cyclic variation (CV) of integrated backscatter. To investigate the effect of intramyocardial contrast on integrated backscatter, we injected 5% sonicated albumin, containing microbubbles in the range of 5 microns in diameter, into the left atrium in nine open-chest dogs. The dogs were anesthetized and placed in the right lateral decubitus position on a specially designed table with a cutout allowing ultrasound imaging from below. Ultrasonic data was obtained from the right precordium by use of a prototype M-mode integrated backscatter system implemented in a commercially available two-dimensional system. Usable data were obtained in eight of nine dogs. Integrated backscatter increased up to 13 dB after contrast injections. There was a significantly decreased CV of integrated backscatter during myocardial contrast in all eight dogs. The mean level of CV of integrated backscatter for the eight dogs decreased from 4.7 dB (530 beats analyzed) without contrast to 2.8 dB during contrast (436 beats analyzed). There was a trend to greater CV at higher levels of contrast. Septal excursion, as measured by M-mode echocardiography simultaneously with integrated backscatter by the same ultrasound beam, was similar with and without contrast (mean 8.2 vs 8.3 mm). Thus left atrium contrast injection produces quantitatively measurable integrated backscatter effects. Cyclic variation of integrated backscatter decreases with contrast. However, at higher contrast levels the decrease tends to be smaller. These effects should be considered during quantitative tissue characterization and myocardial contrast studies.

Albumins↗
PubMed2004

Proteomic analysis of integral plasma membrane proteins.

Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 microg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

Cell Line, Tumor↗
PubMed2004

Signal sequences influence membrane integration of the prion protein.

Biosynthesis of the prion protein at the endoplasmic reticulum generates multiple topological forms. The topology of an individual chain is determined first by the localization of the N terminus and then by potential integration of the transmembrane domain into the lipid bilayer. Here, we provide the first evidence that signal sequences affect the latter of these events by demonstrating that some but not other signal sequences and signal sequence mutations result in significant increases in the fraction of prion protein nascent chains that integrate into the lipid bilayer. Through analysis of the prolactin signal sequence, an especially poor integration effector, we find that the N terminal and hydrophobic regions of the signal sequence affect integration most significantly. Mutations in either region result in a considerable increase in the number of chains that integrate. The effect of the signal sequence cannot be attributed to timing of signal cleavage or the state of the ribosome membrane junction, parameters previously found to affect protein biogenesis. We also present evidence that signal sequences that are poor integration effectors can promote integration under experimental conditions that allow the nascent chain more time to integrate. These findings reveal a previously unappreciated relationship between signal sequences and transmembrane integration.

Amino Acid Sequence↗
PubMed1998

Recording of pacing stimulus artifacts by endovascular defibrillation lead systems: comparison of true and integrated bipolar circuits.

BACKGROUND: The occurrence ICD undersensing of ventricular fibrillation due to the presence of a pacing stimulus artifact (PSA) is in part related to the amplitude of the artifact recorded on the ICD rate sensing circuit. There is little comparative data regarding PSA amplitude recorded by commercial ICD rate-sensing circuits. PURPOSE: To compare PSA amplitude recorded by commercial endovascular defibrillation leads utilizing integrated or true bipolar sensing circuits. METHODS: Nineteen large (60-120 kg) pigs were utilized. Two different commercial endovascular defibrillation leads were evaluated, each with its distal tip located at the right ventricular apex: (1) Medtronic Transvene; and (2) CPI Endotak. Three different rate-sensing circuits were evaluated: (1) Transvene true bipolar (tip-ring); (2) Transvene integrated bipolar (tip-coil); and (3) Endotak integrated bipolar (tip-coil). Using a separate pacing lead located at the left ventricular apex (n = 19 animals) or right ventricular outflow tract (n = 10 animals), pacing was performed at a pulse width of 0.5 milliseconds at outputs of 1.5, 5 and 10 volts. PSA amplitude was recorded at each output by each circuit. RESULTS: During pacing from the left ventricular apex, at each pacing output voltage the PSA amplitude recorded by the true bipolar circuit (0.6 +/- 0.1 mV at 1.5 volts, 2.0 +/- 0.5 mV at 5 volts, 3.7 +/- 0.8 mV at 10 volts) was significantly smaller than recorded by the Transvene integrated circuit (1.4 +/- 0.3 mV at 1.5 volts, 3.8 +/- 0.7 mV at 5 volts, 4.1 +/- 0.8 mV at 10 volts) or the Endotak integrated circuit (1.8 +/- 0.4 mV at 1.5 volts, 4.2 +/- 1.0 mV at 5 volts, 6.3 +/- 1.8 mV at 10 volts). During pacing from the right ventricular outflow tract, at each pacing output voltage the PSA amplitude recorded by the true bipolar circuit (0.7 +/- 0.1 mV at 1.5 volts, 1.7 +/- 0.4 mV at 5 volts, 4.0 +/- 0.7 mV at 10 volts) was significantly smaller than recorded by the Transvene integrated circuit (1.1 +/- 0.4 mV at 1.5 volts, 3.9 +/- 1.2 mV at 5 volts, 7.5 +/- 1.8 mV at 10 volts) or the Endotak integrated circuit (1.6 +/- 0.7 mV at 1.5 volts, 4.3 +/- 1.7 mV at 5 volts, 7.5 +/- 2.6 mV at 10 volts). For both pacing sites, the PSA amplitude recorded by the two integrated circuits was not significantly different. CONCLUSIONS: For a given pacing output voltage, PSA amplitude recorded by commercial endovascular rate sensing/defibrillation leads is greater when the sensing circuit is integrated than when it is true bipolar. These data may be helpful in planning ICD implantation in patients with previously implanted permanent pacemakers.

Animals↗
PubMed2007

Somatic integration from an adenoviral hybrid vector into a hot spot in mouse liver results in persistent transgene expression levels in vivo.

We have developed a hybrid vector that combines the high transduction efficiency of a gene-deleted adenoviral vector and the integration machinery of the bacteriophage-derived integrase PhiC31 for stable transduction and limited integration sites. We based our system on a two-vector system in which the transgene expression cassette is circularized from a helper-dependent vector by Flp-mediated recombination, followed by PhiC31-mediated integration. Integration of the transgene expression cassette from the adenoviral vector resulted in 5-fold higher transgene expression levels in the active PhiC31 group compared to the control group, which received a mutated and inactive version of PhiC31. We confirmed transgene integration into the previously described mpsL1 hot spot of integration by polymerase chain reaction analyses of DNA isolated from mouse livers. In addition, we cloned 40 integration sites. The hot spot mpsL1 was detected only once, and 44% of all integration events were found to be present in gene regions. With further optimization, this system represents a new tool for gene therapy protocols that may offer an alternative to gene therapy approaches based on random integrating viral vectors.

Adenoviridae↗
PubMed1999

Site-specific integration of the conjugal Vibrio cholerae SXT element into prfC.

Vibrio cholerae O139, the first non-O1 serogroup of V. cholerae to give rise to epidemic cholera, is characteristically resistant to the antibiotics sulphamethoxazole, trimethoprim, chloramphenicol and streptomycin. Resistances to these antibiotics are encoded by a 62 kb self-transmissible, conjugative, chromosomally integrating element designated the 'SXT element'. We found that the SXT element integrates site specifically into both V. cholerae and Escherichia coli K-12 into the 5' end of prfC, the gene encoding peptide chain release factor 3. Integration of the SXT element interrupts the chromosomal prfC gene, but the element encodes a new 5' end of prfC that restores the reading frame of this gene. The recombinant of prfC allele created upon element integration is functional. The integration and excision mechanism of the SXT element shares many features with site-specific recombination found in lambdoid phages. First, like lambda, the SXT element forms a circular extrachromosomal intermediate through specific recombination of the left and right ends of the integrated element. Second, chromosomal integration of the element occurs via site-specific recombination in a 17 bp sequence found in the circular form of the SXT element and a similar 17 bp sequence in prfC. Third, both chromosomal integration and excision of the SXT element were found to require an element-encoded int gene with strong similarities to the lambda integrase family. Based on the properties of the SXT element, we propose to classify this element as a CONSTIN, an acronym for a conjugative, self-transmissible, integrating element.

Amino Acid Sequence↗
PubMed2006

Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora.

Homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function. However, integration after transformation is predominantly nonhomologous in most species other than yeast. Here, we show that homologous integration in the filamentous fungus Neurospora requires the homologous-recombination proteins MEI-3 (yeast Rad51 homolog) and MUS-25 (yeast Rad54 homolog), whereas nonhomologous integration requires nonhomologous end-joining protein MUS-52 (yeast Ku80 homolog). Two additional minor integration pathways are present, one MEI-3-independent and homologous, the other MUS-52-independent and nonhomologous. Homologous and nonhomologous mechanisms compete when external DNA is integrated. In Neurospora, both nonhomologous integration pathways, MUS-52-dependent and MUS-52-independent, require MUS-53 (a homolog of human Lig4), which functions in the final step of nonhomologous end-joining. Because nonhomologous integration is eliminated in a LIG4-disrupted strain, integration occurs only at the targeted site in mus-53 mutants, making them an extremely efficient and safe host for gene targeting.

Chromosomes, Fungal↗
PubMed1980

Amplification of integrated viral DNA sequences in polyoma virus-transformed cells.

Polyoma virus (Py) transformation of rat cells requires integration of viral genomes into the host DNA, which generally occurs in a partial or full head-to-tail tandem arrangement. The instability of this structure was previously demonstrated by the high rate of loss of integrated Py genomes in the presence of viral large tumor (T) antigen. We now show that integrated Py DNA sequences can also undergo amplification. We studied two rat cell lines transformed by the ts-a Py mutant, which codes for a thermolabile large T antigen. In a derivative of the ts-a H6A cell line, we have observed loss of full-length Py DNA molecules from the integrated tandem ("curing"), accompanied by the creation of new tandem repeats of two segments of viral DNA corresponding to 38% and 10% of the viral genome, each containing the origin of DNA replication. In the ts-a H3A cell line, which contains an integrated partial tandem of about 1.3 viral genomes with three distinct deletions, propagation at 33 degrees C resulted in the generation of full tandem repeats of a 94% Py DNA "unit" (including two 3% deletions), an 85% "unit" (including a 3% and the 12% deletion), or both. Amplification of integrated viral DNA was not observed in cells propagated at 39.5 degrees C, the nonpermissive temperature for large T antigen function. Amplification of integrated Py DNA sequences thus requires an active large T antigen and can generate a full tandem of integrated viral DNA molecules long after the initial integration event.

Antigens, Neoplasm↗
PubMed1990

Trans-activation function of a 3' truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues.

To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3' end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product.

Amino Acid Sequence↗
PubMed2002

Pros and cons of vertical integration between clinical medicine and basic science within a problem-based undergraduate medical curriculum: examples and experiences from Linköping, Sweden.

Problem-based learning (PBL), combined with early patient contact, multiprofessional education and emphasis on development of communications skills, has become the basis for the medical curriculum at the Faculty of Health Sciences in Linköping (FHS), Sweden, which was started in 1986. Important elements in the curriculum are vertical integration, i.e. integration between the clinical and basic science parts of the curriculum and horizontal integration between different subject areas. This article discusses the importance of vertical integration in an undergraduate medical curriculum, according to experiences from the Faculty of Health Sciences in Linköping, and also give examples on how it has been implemented during the latest 15 years. Results and views put forward in published articles concerning vertical integration within undergraduate medical education are discussed in relation to the experiences in Linköping. Vertical integration between basic sciences and clinical medicine in a PBL setting has been found to stimulate profound rather than superficial learning, and thereby stimulates better understanding of important biomedical principles. Integration probably leads to better retention of knowledge and the ability to apply basic science principles in the appropriate clinical context. Integration throughout the whole curriculum entails a lot of time and work in respect of planning, organization and execution. The teachers have to be deeply involved and enthusiastic and have to cooperate over departmental borders, which may produce positive spin-off effects in teaching and research but also conflicts that have to be resolved. The authors believe vertical integration supports PBL and stimulates deep and lifelong learning.

Biological Science Disciplines↗
PubMed1994

Differential trafficking of soluble and integral membrane secretory granule-associated proteins.

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.

Amino Acid Sequence↗
PubMed1990

Recombination events during integration of transfected DNA into normal human cells.

The mechanisms of recombination responsible for random integration of transfected DNA into the genome of normal human cells have been investigated by analysis of plasmid-cell DNA junctions. Cell clones containing integrated plasmid sequences were selected by morphological transformation of primary human fibroblasts after transfection with a plasmid containing simian virus 40 sequences. Nucleotide sequence analysis of the plasmid-cell DNA junctions was performed on cloned DNA fragments containing the integration sites from two of these cell clones. Polymerase chain reaction was then performed with human cell DNA from primary fibroblasts to isolate the cell DNA from the same sites before plasmid integration. Comparison of the sequences at the plasmid-cell DNA junctions with those of both the original plasmid and the cell DNA demonstrated short sequence similarities and additional nucleotides, typical of nonhomologous recombination. Evidence of short deletions in the cell DNA at the plasmid integration sites suggests that integration occurred by a mechanism similar to that used for repair of spontaneous or gamma ray-induced strand breaks. Plasmid integration occurred within nonrepetitive cell DNA with no major rearrangements, although rearrangements of the cell DNA at the integration site occurred in one of the clones after integration.

Base Sequence↗
PubMed2004

Integration of the 5' end of the retrotransposon, R2Bm, can be complemented by homologous recombination.

R2Bm is a non-long-terminal-repeat (non-LTR) retrotransposon that was identified at a specific target site in the 28S rRNA genes of the silkworm, Bombyx mori. Although in vitro analysis has revealed that the 3' end of R2Bm is integrated into the target site by means of target-primed reverse transcription (TPRT), the mechanism of the 5' end integration is not well understood. We established a novel in vivo system to assay the insertion mechanism of R2Bm using a cultured cell line, C65, and a baculovirus, AcNPV, as host and vector, respectively. The 3' end of R2Bm integrated at the target site in the rRNA genes of C65 cells when an AcNPV containing both the full-length 3' UTR and the entire open reading frame (ORF) of R2Bm was introduced while the 5' end integration was incorrect. The 5' end of R2Bm was integrated, however, when the 28S gene sequence upstream of the R2Bm target site was added to the R2Bm sequence. Thus, in our assay, homologous sequences were likely essential for the successful integration of the entire R2Bm into the host cell genome. We also demonstrated that the failure to integrate caused by a frame-shifted ORF was rescued by co-infection with a helper virus that contained only the R2Bm ORF. This indicates that R2 retrotransposition can be complemented in trans. These findings suggest that the host's mechanism for DNA repair may be necessary for the integration of the 5' end of R2Bm and that R2Bm protein may only have the ability to integrate the 3' end of the element by TPRT.

Animals↗