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L Kleiman

Publications and source records attributed to L Kleiman.

At least 55 records · Page 3Linked to original sources
PubMed1994

Mature reverse transcriptase (p66/p51) is responsible for low levels of viral DNA found in human immunodeficiency virus type 1 (HIV-1).

Reverse transcription of the HIV RNA genome is thought to occur in the host cell cytoplasm after viral adsorption. However, viral DNA has been isolated in cell-free virus particles. We have quantitated by polymerase chain reaction (PCR) amplification the amount of viral DNA in virions as compared to RNA. Virus produced by proviral DNA transfections of cos-7 cells or by chronically-infected H9 cells; neither of which express the cell surface CD4 receptor, contained at least 1000 times more viral RNA than DNA. In contrast, only 60 times more RNA than DNA was present in virus particles produced by transfection of Jurkat cells, which were CD4-positive and thus potentially susceptible to superinfection. Protease-defective virus, carrying only the precursor of reverse transcriptase (RT) p160gag-pol, contained virtually no detectable DNA. These results indicate that only mature RT (p66/p51) and not its precursor (p160gag-pol) is responsible for the presence of viral DNA in HIV.

Cell Line↗
PubMed1993

Expression, purification, and RNA-binding properties of HIV-1 p15gag nucleocapsid protein.

We have cloned and expressed HIV-1 gag p15 nucleocapsid protein (NCp15) in the form of a 41-kDa fusion polypeptide with glutathione-S-transferase (GST-NCp). The recombinant protein was rapidly degraded in bacterial lysates unless Zn2+ and Cd2+ were present in the extraction buffer. Inclusion of these metals stabilized the protein, allowing facile purification of GST-NCp by affinity chromatography. The native NCp15 was readily prepared from GST-NCp by proteolytic cleavage with thrombin. Both GST-NCp and the processed NCp15 were able to bind RNA containing sequences from the 5'-end of the HIV-1 genome. This binding was unaffected by the absence or the presence of Zn2+; however, the binding of RNA was absolutely dependent on the presence of K+. The GST-NCp fusion protein was nonselective in the binding of RNA, with all transcripts, including antisense and non-HIV RNA, binding with equal efficiency. In contrast, NCp15 was highly selective in binding of RNA. Sequences within nucleotides 1244-1412 of the HIV-1 proviral genome were found necessary for maximal binding of RNA to NCp15.

Base Sequence↗
PubMed1993

Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1.

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninfectious HIV-1Lai particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNA(1,2Lys), tRNA(3Lys) (the putative primer for HIV-1 reverse transcriptase) and tRNA(Ile). Identification was accomplished by comparing the electrophoretic mobilities and RNase T1 digests with those of tRNA(3Lys) and tRNA(1,2Lys) purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporation into HIV-1. However, only the wild-type virus contains tRNA(3Lys) tightly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA(3Lys) is based upon an electrophoretic mobility identical to that of tRNA(3Lys) and the ability of this RNA to hybridize with a tRNA(3Lys)-specific DNA probe. In addition to the four wild-type tRNA species, the mutant HIV-1-like particle contains two tRNA(His) species and three tRNA-sized species that we have been unable to identify. Their absence in wild-type virus makes it unlikely that they are required for viral infectivity.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Variable tRNA content in HIV-1IIIB.

Low molecular weight RNA in HIV-1 is found in three size classes resembling 7S RNA, 5S RNA, and tRNA. The 2-dimensional polyacrylamide gel electrophoresis (2D PAGE) patterns of tRNA found in HIV-1 have been determined in virus produced in five different cell types: H9, UHC1 (a U937-derived clone), UHC8 (an RT(-) derivative of U937), HeLa, and COS. The presence of the putative primer tRNA for reverse transcriptase, tRNA(Lys,3), has also been determined either by hybridization with a tRNA(Lys,3)-specific DNA probe or by a comparison of the electrophoretic mobility of viral tRNA species with purified human tRNA(Lys,3). Our results indicate the following: 1) The number of tRNA species found in infectious HIV-1IIIB produced in different cell types varies, according to cell type, from greater than 20 to 4, indicating that only 4 or less tRNA species are required for the viral infectious life cycle. 2) There are 1-3 tRNA species tightly associated to the viral genomic RNA, depending upon the cell type producing the virus. 3) The putative primer tRNA, tRNA(Lys,3), is detected with the tRNA(Lys,3)-specific hybridization probe in the tRNA of HIV-1 produced in H9 cells, and the tightly associated tRNA species in this virus has the same electrophoretic mobility in 1-D PAGE as purified tRNA(Lys,3). On the other hand, we cannot detect tRNA(Lys,3) in the tRNA of HIV-1 produced in HeLa cells, and the tightly associated tRNA found in this virus does not migrate with the same electrophoretic mobility as tRNA(Lys,3).

Animals↗
PubMed1992

Isolation and fractionation of retroviral tRNAs.

Previous studies concerning the analysis of retroviral tRNA populations involved intracellular metabolic labeling of RNA, followed by the isolation of viral RNA and lengthy sucrose gradient centrifugation for the separation of tRNAs found in various viral compartments. A more rapid, convenient, and safer method for achieving similar aims is described. Isolated total viral RNA is end-labeled in vitro, and tRNA subgroups are fractionated using commercial Nucleobond AX-20 mini columns. 2-D PAGE analysis of mouse mammary tumor virus tRNA fractionated in this way yields gel patterns similar to those obtained with previously described methods.

Chromatography↗
PubMed1991

Incorporation of tRNA into normal and mutant HIV-1.

During retroviral assembly, tRNAs are incorporated into the virion, one of which serves as a primer for the reverse transcription reaction. Using two dimensional polyacrylamide gel electrophoresis, we have studied the patterns of tRNAs incorporated into HIV-1 (3B) produced either in the lymphoid cell line H-9 or in the monocytic cell line U937. We have also examined viral tRNA patterns incorporated in a non-infectious, mutant virion which lacks pol gene products and processed gag protein. Our results lead to the following conclusions: 1) tRNA incorporated into HIV-1 is a select subpopulation of the host-cell's tRNA. 2) The type of tRNA incorporated into the virion is dependent upon cell type. 3) There can be multiple species of tRNA of similar mobilities tightly associated to the viral genome. 4) The packaging of putative primer tRNA into virions requires either the synthesis of pol gene products, the processing of gag proteins, or both, while the incorporation of non-primer tRNAs does not.

Cell Line↗
PubMed1991

Cloning, sequencing, and mapping of an alpha-actinin gene from the nematode Caenorhabditis elegans.

The dense-bodies in the body wall muscle of the nematode Caenorhabditis elegans function to anchor the actin thin filaments to the adjacent sarcolemma. One of the major components of the dense-bodies is the actin-binding protein alpha-actinin. To facilitate a genetic analysis of alpha-actinin, we have cloned a cDNA encoding the nematode protein, identified its position on the nematode physical map, and developed a unique PCR based approach to test the position of the cloned gene relative to known genetic deletions. The peptide sequence deduced from the cDNA shows that, apart from a few exceptional regions, the nematode protein shows strong similarity to other known alpha-actinins. Its position on the genetic map shows that none of the known muscle affecting mutations identified in C. elegans are in this alpha-actinin gene. This gene has been given the name atn-1 (alpha-actinin-1).

Actinin↗
PubMed1991

Alterations in cell tetrahydrobiopterin levels may regulate queuine hypomodification of tRNA during differentiation of murine erythroleukemia cells.

The base at the first anticodon ("wobble") position of certain eukaryotic tRNA species is either guanine or the hypermodified base queuine. These tRNA species are synthesized with guanine in the wobble position (tRNAG); this guanine can then be replaced with queuine by the action of the enzyme tRNA-guanine ribosyltransferase. In the present report, we show that tRNAG levels increased in response to the induction of erythroid differentiation of murine erythroleukemia (MEL) cells. We also found that tRNA-guanine ribosyltransferase was significantly inhibited by tetrahydrobiopterin. MEL cells showed a transient threefold increase in tetrahydrobiopterin levels 6 to 12 h after exposure of the cells to inducers such as DMSO or tetramethylurea. The increase in tetrahydrobiopterin preceded the increase in tRNAG which in turn preceded the appearance of phenotypic changes characteristic of differentiation. By contrast, a mutant MEL cell line unable to differentiate in response to inducers showed no change in the level of tetrahydrobiopterin or of tRNAG upon exposure to DMSO. N-acetylserotonin, a well-characterized inhibitor of tetrahydrobiopterin synthesis, prevented the DMSO-mediated increase in tetrahydrobiopterin in normal MEL cells. N-acetylserotonin also inhibited the increase in tRNAG levels and the appearance of phenotypic differentiation in these cells.

Animals↗
PubMed1988

The independent regulation of tRNA(iMet) and tRNA(Asn) synthesis during Friend cell erythroid differentiation.

In this report, we have compared the changes in the production of tRNA(iMet) (initiator tRNA(Met] and tRNA(Asn), which occur during erythroid differentiation in the Friend erythroleukemia cell. The relative steady-state concentration of these two tRNAs (relative to the total tRNA population) was measured by aminoacylation. The results show that while the relative steady-state concentration of tRNA(iMet) changes very little in the cytoplasmic tRNA population, the relative concentration of tRNA(Asn) decreases during the first two days of differentiation and then undergoes an increase. This difference in the behavior of these two tRNAs is also seen when their relative concentrations in newly synthesized tRNA is examined. When tRNA is labeled with tritiated uridine for 24 h in vivo prior to isolation, the hybridization of this labeled tRNA to filter-bound tRNA genes shows that the relative concentration of tRNA(iMet) in newly synthesized tRNA changes very little, while the relative concentration of newly synthesized tRNA(Asn) again decreases through the first 2 days of differentiation, and then undergoes a smaller increase. Thus, the production of these two tRNAs appears to be independently regulated. Independent regulation of synthesis is also observed when examining the production of these two tRNAs in isolated nuclei. During erythroid differentiation, the relative synthesis of tRNA(iMet) (relative to total nuclear RNA synthesis) remains constant, while the relative synthesis of tRNA(Asn) undergoes periodic increases and decreases in value.

Animals↗
PubMed1987

The lateral arm flap: an anatomic study.

The anatomy of the lateral arm flap is further elucidated, and the surgical techniques involved in raising the flap are described in detail. The data are derived from studies in 25 cadavers and five clinical cases. The relative merits of this versatile free flap are discussed. Three clinical cases are cited for illustration.

Amputation, Traumatic↗
PubMed1987

The measurement of the production of tRNA(iMet) during erythroid differentiation of the Friend erythroleukemia cell.

The production of tRNA(iMet) during Friend cell erythroid differentiation has been studied. In vitro measurements of total nuclear RNA synthesis in nuclei isolated from Friend cells at different stages of differentiation show the total RNA synthesis increases 1.5-fold at day 1 of induction and then decreases through days 2 and 3 to approximately 75% of its rate of synthesis in the nuclei of uninduced cells. The synthesis of RNA polymerase III transcripts undergoes a similar fluctuation through day 2 of induction, but increases again at day 3. The specific synthesis of tRNA(iMet) was measured by hybridization of labelled nuclear RNA to a tRNA(iMet) gene probe. During erythroid differentiation the percentage of nuclear RNA represented by tRNA(iMet) remains constant (0.065%), so that the absolute synthesis of tRNA(iMet) fluctuates during differentiation, in the fluctuations in the synthesis of total nuclear RNA. The relative synthesis of tRNA(iMet) in vivo was studied by labelling cells with 32Pi, isolating the resulting radioactive tRNA--5S RNA population, and hybridizing this population to a tRNA(iMet) gene probe. The ratio of tRNA(iMet)/total tRNA--5S RNA in newly synthesized cytoplasmic RNA remains similar throughout differentiation (averaging 0.0171), implying that the fluctuations observed in the nuclear synthesis of tRNA(iMet) during differentiation probably also occur for the nuclear synthesis of most tRNA and 5S RNA species. Attempts were made to measure the relative steady-state concentration of tRNA(iMet) using both aminoacylation and in vitro end labelling of tRNA followed by hybridization to a tRNA(iMet) gene probe. These two methods gave different results and we discuss the possible pitfalls of using enzymatic methods for quantitating tRNA concentrations in the cell.

Animals↗
PubMed1986

The measurement of the production of tRNAMet1 in the Friend erythroleukemia cell.

We have used the gene for tRNAMet1 as a hybridization probe to measure the production of tRNAMet1 in the Friend erythroleukemia cell. In this cell, the relative concentration of tRNAMet1 (i.e., the percentage of total steady-state tRNA representing tRNAMet1) is 1.60 +/- 0.18. To study the relative synthesis of tRNAMet1, cells were labeled in vivo with [3H]uridine for periods ranging from 4 to 24 h, and the tRNA was isolated. The fraction of newly-synthesized tRNA representing tRNAMet1 (1.72% +/- 0.11) does not change when different in vivo labeling times are used. This value is similar to the relative concentration of tRNAMet1 in the older steady-state tRNA (1.61% +/- 0.18). The similar relative synthesis values using different labeling times, plus evidence presented that the total tRNA population decays homogeneously (t 1/2 = 110 h) indicate that tRNAMet1 has a cytoplasmic stability similar to the general tRNA population, and that its concentration relative to the tRNA population is established within the nucleus or soon after exiting the nucleus. Measurements of the synthesis of tRNAMet1 in isolated nuclei, relative to the synthesis of total RNA polymerase III transcripts, showed that this relative synthesis (0.291% +/- 0.017) is only 17% of the relative concentration of tRNAMet1 in the cytoplasm, which may reflect the presence of sequences other than tRNA in total nuclear polymerase III transcripts.

Animals↗
PubMed1984

The use of cloned tRNA genes for the purification and measurement of specific tRNAs.

We have previously reported the ability of a cloned tRNAMeti gene (pt145) to bind tRNAMeti specifically [5]. In this paper, we show that a pBR322 plasmid containing the tRNAAsn gene of Xenopus (pt38 - donated by Stuart Clarkson) will specifically bind to mouse tRNAAsn when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. One-dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band and several small-molecular-weight minor bands. The hybridizing tRNA has been identified as tRNAAsn by partial RNA sequencing and the detection of both the Q base and t6A. The steady-state concentration of tRNAAsn in the uninduced Friend cell was determined by hybridizing tRNA labeled in vitro to pt38. 1% of the total tRNA hybridized, representing 0.017 pg tRNAAsn/cell. The fraction of newly synthesized tRNA representing tRNAAsn or tRNAMeti was also determined by hybridizing tRNA labeled in vivo to either pt38 or pt145, respectively. 0.96% and 0.85% of the tRNA hybridized to pt38 and pt145, respectively.

Animals↗
PubMed1983

The isolation and measurement of tRNAmeti using RNA/DNA hybridization.

A pBR322 plasmid containing the initiator tRNAmet gene of Xenopus (pt145 - donated by Stuart Clarkson) will specifically bind to mouse initiator tRNAmet (tRNAmeti) when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. One dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band (95%) and a minor band. The hybridizing tRNA has been identified as initiator tRNAmet by RNA sequencing. Hybridization of tRNAtotal to another plasmid containing the Xenopus gene for tRNAasn results in two bound species with different electrophoretic mobilities than the tRNA bound to the initiator tRNAmet gene. pt145 has been used to measure the steady state concentration of initiator tRNAmet in the uninduced and erythroid Friend cell, and in the unfertilized egg and 21 h blastula of the sea urchin. Initiator tRNAmet represents 0.91% and 0.52% of the tRNA populations extracted from uninduced and erythroid Friend cells, respectively. Based upon the total tRNA content per cell, there is a 3.8 fold decrease in initiator tRNAmet per cell during erythroid differentiation. tRNA extracted from unfertilized eggs and 21 h blastula of the sea urchin both have 0.5% of total tRNA as initiator tRNAmet (approximately 1.5 pg).

Animals↗
PubMed1978

Alterations in lysine transfer RNA during erythroid differentiation of the Friend cell.

The proportion of lysine tRNA represented by the isoacceptor species lysine tRNA4 has previously been shown to be largest in cells with the greatest ability to proliferate. Using reverse phase chromatography (RPC-5), we have analyzed the changes in the relative quantities of lysine tRNA species which occur in different cellular states of the Friend cell, a transformed murine cell infected with Friend erythroleukemia virus complex. This cell undergoes erythroid differentiation when exposed to various chemicals. Lysine tRNA4 comprises 32% of the total lysine tRNA in rapidly dividing, uninduced Friend cells, but only 16% of the total lysine tRNA in uninducase. Friend cells undergoing erythroid differentiation divide more slowly than uninduced cells, and finally cease proliferation, but lysine tRNA4 becomes the major lysine tRNA species (greater than 50%). This does not appear to reflect erythroid properties of the cell, since the lysine tRNA of the mouse reticulocyte contains very little lysine tRNA4. The non-dividing erythroid Friend cell, therefore, represents an exception to the finding that non-dividing cells usually have little or no lysine tRNA4 present.

Animals↗
PubMed1977

Comparison of the base-sequence complexities of polysomal and nuclear RNAs in growing Friend erythroleukemia cells.

The base-sequence complexities of polysomal poly(A)+ RNA, nuclear poly(A)+ RNA, and total nuclear RNA from Friend erythroleukemia cells in logarithmic phase of growth were determined by measuring the proportion of labeled unique mouse DNA sequences which formed hybrids when incubated with a vast excess of each RNA. It was estimated that the RNAs had been transcribed from 1.8, 7.6, and 8.3%, respectively, of the haploid mouse genome. Although these estimates for polysomal and nuclear poly(A)+ RNAs were 2.5 times greater than those previously determined by analysis of the kinetics of the hybridization reactions between the RNAs and the cDNAs transcribed from them, they confirmed that the base-sequence complexity of nuclear poly(A)+ RNA in these cells is at least four times greater than that of polysomal poly(A)+ RNA.

Base Sequence↗