Biomedical subjects

M A McDevitt

Publications and source records attributed to M A McDevitt.

9 recordsLinked to original sources

PubMed1988

Multiple factors are required for specific RNA cleavage at a poly(A) addition site.

An SP6 RNA containing the adenovirus 5 L3 poly(A) site is processed efficiently in a HeLa cell nuclear extract to generate correct 3' termini. Accurate 3' processing has also been demonstrated for the adenovirus E2A and SV40 early poly(A) sites, although these are processed less efficiently than the L3 site. Efficient cleavage at the poly(A) site requires the presence of a 5'-cap structure, as well as the RNA sequence motifs previously shown to be necessary for 3' processing in vivo, suggesting the presence and action of the appropriate factors in the nuclear extract. Fractionation of the nuclear extract has revealed a requirement for at least two distinct factors for cleavage at the L3 poly(A) site. One of these factors appears to possess an RNA component due to its sensitivity to micrococcal nuclease. The activity of this fraction is also sensitive to alpha-Sm monoclonal antibody, indicating the presence of an snRNP essential for the cleavage reaction. Additional factors are required for the subsequent polyadenylation reaction, indicating the involvement of a multicomponent complex in the processing of an RNA at the poly(A) site.

Adenoviruses, Human↗

PubMed1988

Multiple factors are required for poly(A) addition to a mRNA 3' end.

Polyadenylation of pre-mRNAs in the nucleus involves a specific endonucleolytic cleavage, followed by the addition of approximately 200 adenylic acid residues. We have assayed HeLa nuclear extracts for the activity that catalyzes the poly(A) addition reaction. The authenticity of the in vitro assay was indicated by the observation that the poly(A) tract added in vitro is approximately 200 nucleotides in length. We have fractionated nuclear extracts in order to define components involved in specific poly(A) addition. No single fraction from DEAE-Sephacel chromatography of a HeLa nuclear extract possessed the specific poly(A) addition activity. However, if the various fractions were recombined, activity was restored, indicating the presence of multiple components. Further fractionation revealed the presence of at least two factors necessary for the poly(A) addition reaction. The reconstituted system retains the characteristics and specificity seen in the crude extract. Additional purification of one of the factors strongly suggests it to be a previously characterized poly(A) polymerase which, when assayed in the absence of the other factor, can add AMP to an RNA terminus but without specificity. Thus, the other component of the reaction may provide specificity to the process. In contrast to the 3' cleavage reaction, the poly(A) addition machinery does not possess an essential RNA component, as assayed by micrococcal nuclease digestion, nor do anti-Sm sera inhibit the reaction. Thus, the total process of formation of a polyadenylated mRNA 3' end is complex and requires the concerted action of distinct nuclear components.

Catalysis↗

PubMed1987

Relative position and strengths of poly(A) sites as well as transcription termination are critical to membrane versus secreted mu-chain expression during B-cell development.

During B-cell differentiation, there is a dramatic switch in the RNA products of the immunoglobulin mu heavy chain transcription unit. In the mature B cell there is roughly equal production of the microseconds and the micron RNA, whereas in the antibody-secreting plasma cell there is nearly exclusive production of the microseconds RNA. A plasmid containing the entire mu transcription unit was properly regulated when assayed by transient transfection in a B lymphoma and a plasmacytoma. In contrast, no such regulation was observed with separate plasmids that could produce only one or the other RNA. Instead, the micron poly(A) site was utilized more efficiently than the microseconds poly(A) site, irrespective of the cell type. We also found that transcription termination prior to the micron poly(A) site in plasmacytomas contributes to preferential production of microseconds RNA in these cells. Finally, reducing the distance between the two poly(A) sites improved the use of the micron site at the expense of the use of the microseconds in B lymphoma cells, suggesting a competition for a limiting factor. Such competition was not apparent in plasmacytomas. We conclude that relative poly(A) site strength and the position of the poly(A) sites within the transcription unit, coupled with a changing concentration of a limiting factor, as well as transcription termination prior to the micron poly(A) site, all play a role in determining the expression of the mu locus during B-cell development.

Animals↗

PubMed1986

Sequences capable of restoring poly(A) site function define two distinct downstream elements.

Several recent studies have shown that a functional poly(A) site consists of both an AAUAAA element as well as sequences downstream of the cleavage site. Two downstream regions were analyzed in an attempt to accurately locate and define the critical sequences. Chemically synthesized oligonucleotides of sequence from the early SV40 and the adenovirus E2A poly(A) sites were able to restore efficient cleavage to a deleted SV40 poly(A) site. Inversion of the sequence completely abolished poly(A) site function. A series of base substitution mutants were generated in each downstream sequence. Certain single base changes drastically altered poly(A) site function. Thus, it is concluded that a defined downstream sequence of limited complexity is important for efficient processing of the primary transcript at the poly(A) site. The position of the downstream elements relative to the AAUAAA and cleavage site was found to be critical since moving either the E2 element or the SV40 element an additional 40 nucleotides downstream abolished function. There were differences, however, in the effect of spacing on the function of the two elements. This observation, along with the fact that the two sequences are clearly different, indicates that there are at least two distinct genetic elements that direct efficient cleavage at the poly(A) site.

Adenovirus Early Proteins↗

PubMed1985

Poly(A) site cleavage in a HeLa nuclear extract is dependent on downstream sequences.

Efficient utilization of the early SV40 poly(A) site in vivo requires sequences between 5 bp and 18 bp downstream of the cleavage site. We have used a HeLa nuclear extract to examine the sequence requirements for in vitro cleavage. DNA segments containing the SV40 poly(A) site were cloned into an SP6 vector. SP6 RNAs, accurately cleaved and polyadenylated, were detected by primer extension. Cleavage was enhanced by the presence of a cap on the primary transcript, and was inhibited by the addition of 10 microM 7meGpppG. In close agreement with the in vivo results, efficient processing at the poly(A) site in vitro required the specific downstream sequences in the SP6 RNA transcript. These experiments indicate that the sequence in the RNA precursor downstream of the cleavage site, shown to be important for efficient processing in vivo, is recognized in vitro.

Base Sequence↗

PubMed1985

Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites.

In addition to the highly conserved AATAAA sequence, there is a requirement for specific sequences downstream of polyadenylic acid [poly(A)] cleavage sites to generate correct mRNA 3' termini. Previous experiments demonstrated that 35 nucleotides downstream of the E2A poly(A) site were sufficient but 20 nucleotides were not. The construction and assay of bidirectional deletion mutants in the adenovirus E2A poly(A) site indicates that there may be redundant multiple sequence elements that affect poly(A) site usage. Sequences between the poly(A) site and 31 nucleotides downstream were not essential for efficient cleavage. Further deletion downstream (3' to +31) abolished efficient cleavage in certain constructions but not all. Between +20 and +38 the sequence T(A/G)TTTTT was duplicated. Function was retained when one copy of the sequence was present, suggesting that this sequence represents an essential element. There may also be additional sequences distal to +43 that can function. To establish common features of poly(A) sites, we also analyzed the early simian virus 40 (SV40) poly(A) site for essential sequences. An SV40 poly(A) site deletion that retained 18 nucleotides downstream of the cleavage site was fully functional while one that retained 5 nucleotides downstream was not, thus defining sequences required for cleavage. Comparison of the SV40 sequences with those from E2A did not reveal significant homologies. Nevertheless, normal cleavage and polyadenylation could be restored at the early SV40 poly(A) site by the addition of downstream sequences from the adenovirus E2A poly(A) site to the SV40 +5 mutant. The same sequences that were required in the E2A site for efficient cleavage also restored activity to the SV40 poly(A) site.

Adenovirus Early Proteins↗

PubMed1984

Malic enzyme and fatty acid synthase in the uropygial gland and liver of embryonic and neonatal ducklings. Tissue-specific regulation of gene expression.

Malic enzyme [L-malate-NADP oxidoreductase (decarboxylating), EC 1.1.1.40] and fatty acid synthase activities were barely detectable in the uropygial gland of duck embryos until 4 or 5 days before hatching, when they began to increase. These activities increased about 30- and 140-fold, respectively, by the day of hatching. Malic enzyme and fatty acid synthase activities were also very low in embryonic liver. However, hepatic malic enzyme activity did not increase until the newly hatched ducklings were fed. Hepatic fatty acid synthase began to increase the day before hatching and the rate of increase in enzyme activity accelerated markedly when the newly hatched ducklings were fed. Starvation of newly hatched or 12-day-old ducklings had no effect on the activities of malic enzyme and fatty acid synthase in the uropygial gland but markedly inhibited these activities in liver. Changes in the concentrations of both enzymes and in the relative synthesis rates of fatty acid synthase correlated with enzyme activities in both uropygial gland and liver. Developmental patterns for sequence abundance of malic enzyme and fatty acid synthase mRNAs in uropygial gland and liver were similar to those for their respective enzyme activities. Starvation of 4-day-old ducklings had no significant effect on the abundance of these mRNAs in uropygial gland but caused a pronounced decrease in their abundance in liver. It is concluded that developmental and nutritional regulation of these enzymes is tissue specific and occurs primarily at a pretranslational level in both uropygial gland and liver.

Animals↗

PubMed1984

Requirement of a downstream sequence for generation of a poly(A) addition site.

The 3' terminus of most, if not all, eucaryotic polyadenylated mRNAs is formed as a result of endonucleolytic cleavage of a larger precursor RNA. That is, transcription does not terminate at the mRNA 3' sequence but rather proceeds through this site, terminating at some distance downstream. Using a plasmid containing the adenovirus E2A transcriptional unit, we have investigated the sequence requirement for the formation of a mature mRNA 3' terminus, focusing on the role of sequences immediately distal to the poly(A) addition site. Deletion mutants were constructed in the region distal to the poly(A) addition site and assayed by transfection into human 293 cells. The results demonstrate that 35 nucleotides distal to the site of poly(A) addition are sufficient for the formation of a mature E2 mRNA. However, removal of an additional 15 nucleotides, leaving 20 nucleotides distal to the poly(A) site, abolished the ability to produce functional E2A mRNA. The defect in the production of functional mRNA from such a mutant appears to be in the proper cleavage of the primary transcript at the poly(A) addition site. It would thus appear that sequences immediately distal to the site of poly(A) addition do not contribute to the mature mRNA but are essential for the formation of mature mRNA.

Adenoviruses, Human↗

PubMed1982

Molecular cloning of gene sequences for avian fatty acid synthase and evidence for nutritional regulation of fatty acid synthase mRNA concentration.

A double-stranded cDNA library was constructed using total poly(A)+ RNA from the goose uropygial gland. Clones containing sequences complementary to fatty acid synthase mRNA were initially identified by colony hybridization with a 32P-labeled cDNA transcribed from RNA enriched for fatty acid synthase mRNA. Identity of the fatty acid synthase clones was confirmed by hybrid-selected translation. Mature fatty acid synthase mRNA is approximately 16 kilobases in length. When unfed neonatal goslings were fed for 24 hr, relative synthesis of hepatic fatty acid synthase increased more than 42-fold. Concomitantly, hepatic fatty acid synthase mRNA levels increased 70-fold. Thus, nutritional regulation of the synthesis of hepatic fatty acid synthase probably occurs at the pretranslational level. The availability of a specific probe for fatty acid synthase mRNA should allow us to analyze the regulation of expression of this gene during development, by nutrition and by hormones in both liver and uropygial gland.

Animals↗