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S Connelly

Publications and source records attributed to S Connelly.

36 records · Page 2Linked to original sources
PubMed1996

Sustained expression of therapeutic levels of human factor VIII in mice.

Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression.

Adenoviruses, Human↗
PubMed1996

High-level tissue-specific expression of functional human factor VIII in mice.

Hemophilia A results from subnormal levels of blood coagulation factor VIII (FVIII) and is an attractive target for gene therapy. However, progress has been impeded by features of FVIII biology such as low mRNA accumulation and the instability of the protein. We have shown previously that a FVIII adenoviral vector, Av1ALH81, allowed high-level expression of human FVIII in mice sustained for several weeks. Here, we have generated a second FVIII adenoviral vector, Av1ALAPH81, in which an intron was introduced into the FVIII expression cassette. Administration of Av1ALAPH81 to mice resulted in significantly increased FVIII plasma levels, 1,046 +/- 163 ng/ml compared to 307 +/- 93 ng/ml of FVIII detected in mice that received Av1ALH81. Normal FVIII levels in humans are 100-200 ng/ml and therapeutic levels are as low as 10 ng/ml. Therapeutic levels are defined as the amount of FVIII necessary to convert severe hemophilia to a moderate or mild hemophiliac condition. The increased potency of the second FVIII adenoviral vector allowed the administration of significantly lower, less toxic vector doses, while retaining the potential for high FVIII expression. Furthermore, we demonstrate that adenoviral-mediated expression of human FVIII can be limited to the liver by inclusion of a liver-specific promoter, thereby achieving the first step in regulated expression of human FVIII in vivo.

Adenoviruses, Human↗
PubMed1995

In vivo gene delivery and expression of physiological levels of functional human factor VIII in mice.

Hemophilia A is caused by blood coagulation factor VIII (FVIII) deficiency and is an attractive target for gene therapy. However, features of FVIII physiology, such as the instability of the mRNA and protein, have provided obstacles to the design of a feasible strategy for the transfer and expression of the human FVIII gene in vivo. We have constructed a recombinant adenoviral vector, Av1ALH81, that contains the human FVIII cDNA from which the B-domain has been deleted (BDD FVIII) and extensively characterized this vector in vitro and in vivo. In vitro, HepG2, human hepatoma cells, transduced with Av1ALH81 secreted high levels of biologically active human BDD FVIII measured by the Coatest bioassay (> 2,400 mU per 10(6) cells per 24 hr). Administration of Av1ALH81 to mice, via tail vein, resulted in expression of human BDD FVIII in the mouse plasma at levels averaging 307 +/- 93 ng/ml 1 week post-injection, measured by a sensitive human FVIII-specific ELISA. Normal FVIII levels in humans are 100-200 ng/ml, and therapeutic levels are as low as 10 ng/ml. Purification of the human FVIII from the mouse plasma, and subsequent Coatest analysis, revealed that the human FVIII produced in the mice was biologically active. In addition, the duration of FVIII expression in vivo was followed, and high-level FVIII expression was sustained over a period of several weeks. The finding that an adenoviral vector can mediate high-level expression of human FVIII in an animal model provides the basis for the development of gene therapy for hemophilia A.

Adenoviridae↗
PubMed1994

Characterisation and expression of a maize U3 snRNA gene.

We have used a probe encoding a U3snRNA gene of Arabidopsis to isolate maize U3snRNA genomic sequences. Of two clones sequenced, one encodes a single U3 gene which has been shown to be expressed in transfected maize protoplasts. The second clone encodes a U3 related sequence which appears to be an RNA-mediated pseudogene.

Arabidopsis↗
PubMed1994

Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts.

RNA polymerase (Pol) II- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledonous plants contain two essential upstream promoter elements, the USE and TATA. The USE is a highly conserved plant snRNA gene-specific element, and its distance from the -30 TATA box, corresponding to approximately three and four helical DNA turns in Pol III and Pol II genes, respectively, is crucial for determining RNA Pol specificity of transcription. Sequences upstream of the USE play no role in snRNA gene transcription in dicot plants. Here we show that for expression of snRNA genes in maize, a monocotyledonous plant, the USE and TATA elements are essential, but not sufficient, for transcription. Efficient expression of both Pol II- and Pol III-specific snRNA genes in transfected maize protoplasts requires an additional element(s) positioned upstream of the USE. This element, named MSP (for monocot-specific promoter; consensus, RGCCCR), is present in one to three copies in monocot snRNA genes and is interchangeable between Pol II- and Pol III-specific genes. The efficiency of snRNA gene expression in maize protoplast is determined primarily by the strength of the MSP element(s); this contrasts with the situation in protoplasts of a dicot plant, Nicotiana plumbaginifolia, where promoter strength is a function of the quality of the USE element. Interestingly, the organization of monocot Pol III-specific snRNA gene promoters closely resembles those of equivalent vertebrate promoters. The data are discussed in the context of the coevolution of Pol II- and Pol III-specific snRNA gene promoters within many eukaryotic organisms.

Arabidopsis↗
PubMed1993

Differential expression of U5snRNA gene variants in maize (Zea mays) protoplasts.

The small nuclear ribonucleoprotein particles U1, U2, U4/U6 and U5 participate in the removal of introns from pre-messenger RNAs in the nucleus. Three genes encoding U5snRNAs, the RNA moiety of U5snRNPs, have been isolated from maize. As in other plant UsnRNA gene families the three maize U5snRNA genes exhibit sequence variation. Two of the gene variants (MzU5.1 and MzU5.2) are clearly expressed after transfection into maize leaf protoplasts while the third gene variant (MzU5.3) is expressed at very low levels. These different levels of expression cannot be directly correlated with sequence changes in the highly conserved Upstream Sequence Element (USE) required for expression of Arabidopsis UsnRNA genes nor with differential stability of the U5snRNA transcripts. Further sequence elements may therefore have a role in regulating maize UsnRNA gene expression.

Base Sequence↗
PubMed1993

Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants.

Formation of the 3' ends of RNA polymerase II (Pol II)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plumbaginifolia. We found that the 3'-end-adjacent sequence CA (N)3-10AGTNNAA, conserved in plant Pol II-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end formation signal, indicating that these signals are functionally equivalent among different Pol II-transcribed snRNA genes. The plant U snRNA 3'-end formation signal can be recognized irrespective of whether transcription initiation occurs at U snRNA or mRNA gene promoters, although efficiency of 3' box utilization is higher when transcription initiation occurs at the U snRNA promoter. Moreover, transcripts initiated from the U2 gene promoter can be spliced and polyadenylated. Transcription from a Pol III-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol II-specific plant U snRNA gene promoter can occur in the absence of the snRNA coding region and a functional snRNA 3'-end formation signal, demonstrating that these sequences play no role in determining the RNA polymerase specificity of plant U snRNA genes.

Arabidopsis↗
PubMed1993

Talonavicular coalition. Objective gait analysis.

Talonavicular coalitions, a rarely reported fusion between the talus and navicular, are often an incidental radiographic finding that may be asymptomatic or associated with peroneal spasm. The authors present a review of literature and case report based on clinical evaluation and instrumented gait analysis. Specifically, a patient presenting with a bilateral talonavicular coalition was objectively evaluated with kinetic, kinematic, muscle dynamometry, and pedobarographic testing to understand the biomechanical limitations related to this pathology. An excessive passive component of ankle torque, a high first metatarsophangeal joint plantar pressure, and a diminished time in the midstance portion of stance phase were measured and compared to those of healthy individuals.

Adolescent↗
PubMed1992

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNA polymerase III.

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3'-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to alpha-amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.

Amanitins↗
PubMed1991

Complex health problems. Clinically assessing self-management abilities.

Assessment of self-care abilities by staff nurses is critical to planning post-hospitalization care. Assessment instruments must be comprehensive and yet usable by busy nurses. Orem's three categories of self-care deficits (universal, developmental, and therapeutic) provided a basis for the development of a Self-Management Inventory.

Aged↗
PubMed1989

A CCAAT box sequence in the adenovirus major late promoter functions as part of an RNA polymerase II termination signal.

Termination of transcription by RNA polymerase II has been shown in several cases to require a functional poly(A) addition site, although the actual termination event occurs further downstream. To define in more detail the sequences required for termination, we mapped the site at which transcription terminates on a chimeric plasmid template that contains the adenovirus MLP directing transcription of the SV40 early region. Termination in cells transfected with this plasmid occurs within a discrete promoter-proximal region that contains an inverted CCAAT-box sequence. This region of the MLP was also capable of directing termination, in an orientation-dependent manner, when inserted downstream of the poly(A) site in the plasmid template. In addition, in adenovirus-infected cells, transcription initiated from upstream promoters on the adenovirus chromosome terminates within the same MLP promoter-proximal region, both establishing the physiological relevance of the observed CCAAT-box dependent termination, and also suggesting a possible function for transcription termination in adenovirus infection.

Adenoviridae↗
PubMed1989

RNA polymerase II transcription termination is mediated specifically by protein binding to a CCAAT box sequence.

A region in the adenovirus major late promoter (MLP) containing a CCAAT consensus sequence can direct transcription termination of RNA polymerase II, a mechanism that possibly prevents transcriptional interference from upstream genes. Using a chimeric plasmid template that contains the MLP directing expression of the simian virus 40 early region, we showed that an inserted oligonucleotide containing only 13 base pairs of MLP sequences, including the CCAAT box, is capable of inducing transcription termination in an orientation-dependent, position-independent manner. Point mutations within the CCAAT-specific protein-binding site abolished this effect, while a base substitution outside of this region did not affect termination. These data suggest that termination is mediated by a CCAAT box-binding protein. Several other transcription factor-binding sites do not, however, cause termination, suggesting that this may be a relatively specific property of a CCAAT-binding protein.

Adenoviruses, Human↗
PubMed1988

A functional mRNA polyadenylation signal is required for transcription termination by RNA polymerase II.

Polyadenylation of pre-mRNAs requires the conserved hexanucleotide AAUAAA, as well as sequences located downstream from the poly(A) addition site. The role of these sequences in the production of functional mRNAs was studied by analyzing a series of mutants containing deletions or substitutions in the SV40 early region poly(A) site. As expected, both a previously defined GU-rich downstream element and an AAUAAA sequence were required for efficient usage of the wild-type poly(A) addition site. However, when either of these elements was deleted, greatly increased levels of SV40-specific RNA were detected in the nuclei of transfected cells. Evidence is presented that this accumulation of RNA resulted from a failure of transcription termination, leading to multiple rounds of transcription of the circular templates. We conclude that the sequences required for efficient cleavage/polyadenylation of the SV40 early pre-mRNA also constitute an important element of an RNA polymerase II termination signal. A model proposing a mechanism by which the act of pre-mRNA 3' end formation is signaled to the elongating RNA polymerase, resulting in termination, is presented.

Base Sequence↗
PubMed1985

Identification of a sequence element on the 3' side of AAUAAA which is necessary for simian virus 40 late mRNA 3'-end processing.

Our previous studies of the 3'-end processing of simian virus 40 late mRNAs indicated the existence of an essential element (or elements) downstream of the AAUAAA signal. We report here the use of transient expression analysis to study a functional element which we located within the sequence AGGUUUUUU, beginning 59 nucleotides downstream of the recognized signal AAUAAA. Deletion of this element resulted in (i) at least a 75% drop in 3'-end processing at the normal site and (ii) appearance of readthrough transcripts with alternate 3' ends. Some flexibility in the downstream position of this element relative to the AAUAAA was noted by deletion analysis. Using computer sequence comparison, we located homologous regions within downstream sequences of other genes, suggesting a generalized sequence element. In addition, specific complementarity is noted between the downstream element and U4 RNA. The possibility that this complementarity could participate in 3'-end site selection is discussed.

Base Sequence↗
PubMed1979

Tendon transfers in the paralytic hip.

We analyzed the results of transfers of the iliopsoas or external oblique muscles performed to augment the abductor power of the hips in 149 patients with flaccid paralysis of the hips. More than 800 supplemental procedures were performed on the lower extremities of these patients. Twenty-three of the thirty-eight hips that had been subluxated or dislocated became stable in the reduced position after the transfer operation. Every hip that had been stable before the transfer remained stable, and thirty-seven patients who had been brace-dependent became brace-free. We concluded that transfers of the iliopsoas or external oblique muscles are useful adjuncts in the treatment of paralytic disorders of the hips.

Adolescent↗