Jim Karam - Academia.edu (original) (raw)

Papers by Jim Karam

Blood, Jul 1, 1990

We examined "y-globin-chain biosynthesis by adult and umbilical cord blood erythropoietic bursts ... more We examined "y-globin-chain biosynthesis by adult and umbilical cord blood erythropoietic bursts in methylcellulose clonal culture and 'y-chain synthesis by cord blood reticulocytes. Globin chains were labeled with '4C-amino acids and quantitated by using autoradiography or fluorography. Alpha, beta, and G'y and A'y chains were separated by isoelectric focusing in polyacrylamide gels containing 8 M urea and 3% Nonidet P-40 (a nonionic detergent). Time course examinations of the 'y-chains synthesized by the bursts revealed no changes in the Gy:Ay ratio between days 1 0 and 1 8 of culture. The ratio of G"y/(G'y + ky) in cultures of adult circulating erythroid precursors was 0.38 ± 0.09, which corresponds to the known ratio in adult peripheral blood erythrocytes. The relative &'y-chain biosyntheses in the cord blood bursts and reticulocytes were

Genetics, 2002

Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated p... more Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3′ exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol+ Exo+) enzyme, an exonuclease-deficient mutator variant (Pol+ Exo-), mutator variants with substitutions at Tyr567 in the polymerase active site (PolM Exo+), and the double mutator PolM Exo-. Comparing the mutational spectra of the Pol+ Exo- and Pol+ Exo+ enzymes revealed the patterns and efficiencies of proofreading, while Tyr567 was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat differe...

Blood, 1980

By using a methylcellulose clonal assay, we cultured peripheral blood erythropoietic precursors (... more By using a methylcellulose clonal assay, we cultured peripheral blood erythropoietic precursors (BFU-E) from an adult couple whose child had HbF Malta-I(gamma 117 His leads to Arg), a G gamma variant, and measured the synthetic rates of HbA, HbF, and HbF Malta-I. Hemoglobin was labeled with 14C-amino acid in culture, separated by slab gel isoelectric focusing technique, and quantitated by autoradiographic or fluorographic method. Culture of BFU-E from both parents revealed significant HbF biosynthesis. HbF Malta-I was present in culture of the father's cells and comprised about 24% of total HbF. When we analyzed Hb biosynthesis in individual bursts, all bursts contained HbA and HbF in varying ratios. The frequency distribution of the individual bursts differing in percentages of HbF biosynthesis approached normal distribution. While the relative ratio of HbF Malta-I to total HbF biosynthesis in individual bursts also revealed significant variation, its frequency distribution did...

Journal of Molecular Biology, 2010

Virology journal, Jan 23, 2006

Bacteriophages are an important repository of genetic diversity. As one of the major constituents... more Bacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. The contribution of the T4-like phages to this overall phage diversity is difficult to assess, since only a few examples of complete genome sequence exist for these phages. Our analysis of five T4-like genomes represents half of the known T4-like genomes in GenBank. Here, we have examined in detail the genetic diversity of the genomes of five relatives of bacteriophage T4: the Escherichia coli phages RB43, RB49 and RB69, the Aeromonas salmonicida phage 44RR2.8t (or 44RR) and the Aeromonas hydrophila phage Aeh1. Our data define a core set of conserved...

Virology journal, Jan 17, 2004

The single-strand binding (Ssb) protein of phage T4 (T4 gp32, product of gene 32) is a mRNA-speci... more The single-strand binding (Ssb) protein of phage T4 (T4 gp32, product of gene 32) is a mRNA-specific autogenous translational repressor, in addition to being a sequence-independent ssDNA-binding protein that participates in phage DNA replication, repair and recombination. It is not clear how this physiologically essential protein distinguishes between specific RNA and nonspecific nucleic acid targets. Here, we present phylogenetic evidence suggesting that ssDNA and specific RNA bind the same gp32 domain and that plasticity of this domain underlies its ability to configure certain RNA structures for specific binding. We have cloned and characterized gene 32 of phage RB69, a relative of T4 We observed that RB69 gp32 and T4 gp32 have nearly identical ssDNA binding domains, but diverge in their C-terminal domains. In T4 gp32, it is known that the C-terminal domain interacts with the ssDNA-binding domain and with other phage-induced proteins. In translation assays, we show that RB69 gp32...

We examined &amp;amp;quot;y-globin-chain biosynthesis by adult and umbilical cord blood e... more We examined &amp;amp;quot;y-globin-chain biosynthesis by adult and umbilical cord blood erythropoietic bursts in methylcellulose clonal culture and &amp;amp;#39;y-chain synthesis by cord blood reticulocytes. Globin chains were labeled with &amp;amp;#39;4C-amino acids and quantitated by using autoradiography or fluorography. Alpha, beta, and G&amp;amp;#39;y and A&amp;amp;#39;y chains were separated by isoelectric focusing in polyacrylamide gels containing 8 M urea and 3% Nonidet P-40

Journal of Biological Chemistry, 1997

DNA polymerase of phage T4 (T4 gp43), an essential component of the T4 DNA replicase, is a multif... more DNA polymerase of phage T4 (T4 gp43), an essential component of the T4 DNA replicase, is a multifunctional single-chained (898-amino acid) protein that catalyzes the highly accurate synthesis of DNA in phage replication. The enzyme functions both as a DNA-binding replication protein and as a sequence-specific RNA-binding autogenous translational repressor. We have utilized a phylogenetic approach to study the relationships between the two nucleic acid-binding functions of the protein. We found that autogenous translational control of gp43 biosynthesis has been conserved in phage RB69, a distant relative of T4, although we also found that the RB69 system differs from its T4 counterpart in two regards: (a) nucleotide sequence and predicted secondary structure of the RNA target (translational operator), and (b) RNA specificity of the protein. T4 gp43 is specific to the RNA operator sequence of the T4 genome whereas RB69 gp43 can bind and repress operator RNA from both phages equally well. In studies with T4-RB69 gp43 chimeras, we mapped T4 gp43 RNA-binding specificity to a protein segment that also harbors important determinants for DNA binding and the polymerase catalytic function. Our results suggest that RNA functions as a regulator of both the dosage and activity of this DNA replication enzyme.

Journal of Biological Chemistry, 1995

We describe the use of a phylogenetic approach to analyze the modular organization of the single-... more We describe the use of a phylogenetic approach to analyze the modular organization of the single-chained (898 amino acids) and multifunctional DNA polymerase of phage T4. We have identified, cloned in expression vectors, and sequenced the DNA polymerase gene (gene 43) of phage RB69, a distant relative of T4. The deduced primary structure of the RB69 protein (RB69 gp43) differs from that of T4 gp43 in discrete clusters of short sequence that are interspersed with clusters of high similarity between the two proteins. Despite these differences, the two enzymes can substitute for each other in phage DNA replication, although T4 gp43 does exhibit preference to its own genome. A 55-amino acid internal gp43 segment of high sequence divergence between T4 and RB69 could be replaced in RB69 gp43 with the corresponding segment from T4 without loss of replication function. The reciprocal chimera and a deletion mutant of the T4 gp43 segment were both inactive for replication and specifically inhibitory ("dominant lethal") to the T4 wild-type allele. The results show that phylogenetic markers can be used to construct chimeric and truncated forms of gp43 that, although inactive for replication, can still exhibit biological specificity.

Journal of Biological Chemistry, 2002

DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding rep... more DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding replication enzyme and the other as an mRNA-specific autogenous translational repressor. Binding of T4 gp43 to its mRNA target (translational operator RNA) interferes with gp43-DNA interactions, but it is unclear how the protein determinants for binding DNA are affected by the dynamics of gp43-mRNA interactions. We have used RB69 gp43, a natural variant of the T4 enzyme whose crystal structure has been determined to identify protein sites that respond to the interaction with specific RNA. We used protein phosphorylation markers, photocross-linking studies, protease sensitivity assays, and mutational analyses to examine the effects of operator RNA on the enzyme's five structural domains (N, exo, palm, fingers, and thumb). Our studies suggest that this RNA affects gp43-DNA interactions through global effects on protein structure that occlude DNA-binding sites but leave the enzyme accessible to interactions with the sliding clamp (RB69 gp45) and possibly other polymerase accessory proteins. We discuss the possible biological significance of putative RNA-binding motifs in the N and palm domains of RB69 gp43.

Journal of Biological Chemistry, 2000

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase ␣ ... more The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase ␣ class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo ؊ enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr 567 (Pol Y567(A/S/T) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr 567 is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the Pol Y567A Exo ؊ enzyme generates mispairs more frequently but extends them less efficiently than does a Pol ؉ Exo ؊ enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43.

Hemoglobin, 1978

We cultured human umbilical cord blood and adult peripheral blood erythropoietic precursors in me... more We cultured human umbilical cord blood and adult peripheral blood erythropoietic precursors in methylcellulose clonal assay and measured the synthetic rates of HbA, A2, and F. Hb was labeled with 14C-amino acid in culture and separated by slab-gel isoelectric focusing and quantitated by autoradiography. While the mean percentage of HbF synthesized by adult cells was only 20.1%, that of umbilical cord blood cells was 53.9%, which corresponds closely to the biosynthetic capabilities of umbilical cord blood reticulocytes. Variations in the erythropoietin concentrations did not influence the percentage of HbF. Erythropoietic cell cultures of human umbilical cord blood may provide an important means for studying the molecular mechanisms controlling physiological Hb switching in the perinatal period.

Cell, 1997

amino acid sequence of the bacteriophage RB69 DNA and T. A. Steitz* † ‡ polymerase (gp43), whose ... more amino acid sequence of the bacteriophage RB69 DNA and T. A. Steitz* † ‡ polymerase (gp43), whose structure is reported here, is * Department of Molecular Biophysics and 63% identical to that of its homolog from T4 phage and Biochemistry is similar to the primary structures of several pol ␣ family † Department of Chemistry DNA polymerases (Wang et al., 1995). ‡ Howard Hughes Medical Institute The structures of the E. coli Klenow fragment (KF) and Yale University Thermus aquaticus DNA polymerase (Taq pol) from the New Haven, Connecticut 06520-8114 pol I family (Ollis et al., 1985; Kim et al., 1995) and that § School of Medicine of the HIV reverse transcriptase (RT) (Kohlstaedt et al., Department of Biochemistry 1992) and their DNA complexes (Beese et al., 1993b; Tulane University Jacobo-Molina et al., 1993; Eom et al., 1996) show a New Orleans, Louisiana 70112 U-shaped polymerase domain geometry that has been likened to the shape of a hand with "thumb," "palm," and "fingers" subdomains (Ollis et al., 1985; Kohlstaedt Summary et al., 1992). The thumb interacts across the minor groove of product duplex DNA, while the palm contains The 2.8 Å resolution crystal structure of the bacteriothe polymerase catalytic site; the fingers interact with phage RB69 gp43, a member of the eukaryotic pol ␣ the template strand and perhaps with the deoxynucleofamily of replicative DNA polymerases, shares some side triphosphate. While the structure of mammalian similarities with other polymerases but shows many DNA polymerase ␤ (pol ␤) exhibits an analogous handdifferences. Although its palm domain has the same like shape (Davies et al., 1994; Pelletier et al., 1994; topology as other polymerases, except rat DNA poly-Sawaya et al., 1994), it is not structurally homologous merase ␤, one of the three carboxylates required for to pol I, RT, or T7 RNA polymerase (T7 RNAP) (Steitz et nucleotidyl transfer is located on a different ␤ strand. al., 1994) but rather belongs to another family of nucleoti-The structures of the fingers and thumb domains are dyl transferases (Holm and Sander, 1995; Yue et al., unrelated to all other known polymerase structures. 1996). The editing 3-5 exonuclease domain of gp43 is ho-The structures of the polymerase domains of pol I, mologous to that of E. coli DNA polymerase I but lies RT, and T7 RNAP (Ollis et al., 1985; Kohlstaedt et al., on the opposite side of the polymerase active site. 1992; Jacobo-Molina et al., 1993; Sousa et al., 1993) An extended structure-based alignment of eukaryotic show a surprising amount of diversity, although the cata-DNA polymerase sequences provides structural inlytic palm domains of all three are sufficiently similar to sights that should be applicable to most eukaryotic imply homology. In contrast, their thumb-domain struc-DNA polymerases. tures are not alike, and the structures of only the pol I and RNA pol fingers domains are similar. Amino acid

Journal of Molecular Biology, 2006

We have completely sequenced and annotated the genomes of several relatives of the bacteriophage ... more We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter johnsonii phage 133 and Vibrio natriegens phage nt-1. Each of these phage genomes exhibited a unique sequence that distinguished it from its relatives, although there were examples of genomes that are very similar to each other. As a group the phages compared here diverge from one another by several criteria, including (a) host range, (b) genome size in the range between ∼160 kb and ∼250 kb, (c) content and genetic organization of their T4-like genes for DNA metabolism, (d) mutational drift of the predicted T4-like gene products and their regulatory sites and (e) content of open-reading frames that have no counterparts in T4 or other known organisms (novel ORFs). We have observed a number of DNA rearrangements of the T4 genome type, some exhibiting proximity to putative homing endonuclease genes. Also, we cite and discuss examples of sequence divergence in the predicted sites for protein-protein and protein-nucleic acid interactions of homologues of the T4 DNA replication proteins, with emphasis on the diversity in sequence, molecular form and regulation of the phageencoded DNA polymerase, gp43. Five of the sequenced phage genomes are predicted to encode split forms of this polymerase. Our studies suggest that the modular construction and plasticity of the T4 genome type and several of its replication proteins may offer resilience to mutation, including DNA rearrangements, and facilitate the adaptation of T4-like phages to different bacterial hosts in nature.

Journal of Bacteriology, 1998

The genomes of bacteriophages T4 and RB69 are phylogenetically related but diverge in nucleotide ... more The genomes of bacteriophages T4 and RB69 are phylogenetically related but diverge in nucleotide sequence at many loci and are incompatible with each other in vivo. We describe here the biological implications of divergence in a genomic segment that encodes four essential DNA replication proteins: gp45 (sliding clamp), gp44/62 complex (clamp loader), and gp46 (a recombination protein). We have cloned, sequenced, and expressed several overlapping segments of the RB69 gene 46-45.2-(rpbA)-45-44-62 cluster and compared its features to those of the homologous gene cluster from T4. The deduced primary structures of all four RB69 replication proteins and gp45.2 from this cluster are very similar (80 to 95% similarity) to those of their respective T4 homologs. In contrast, the rpbA region (which encodes a nonessential protein in T4) is highly diverged (approximately 49% similarity) between the two phage genomes and does not encode protein in RB69. Expression studies and patterns of high div...

which permits unrestricted use, distribution, and reproduction in any medium, provided the origin... more which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Bacteriophage research continues to break new ground in our understanding of the basic molecular mechanisms of gene action and biological structure. The abundance of bacteriophages in nature and the diversity of their genomes are two reasons why phage research brims with excitement. The pages of Virology Journal will reflect the excitement of the "New Phage Biology." The launching of Virology Journal comes at a time of resurgence of interest in the basic biology of the bacteriophages and the impact that these viruses have on earth's ecology, evolution of microbial diversity and the control of infectious disease. Since playing an important part in the birth of Molecular Biology more than 50 years ago [1], phage research has continually broken new ground in our understanding of the basic molecular mechanisms of gene action and biological structure ...

© 2004 Borjac-Natour et al; licensee BioMed Central Ltd. This is an open-access article distribut... more © 2004 Borjac-Natour et al; licensee BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License

Interaction of Translational and Transcriptional Controls in the Regulation of Gene Expression, 1982

Progress in Nucleic Acid Research and Molecular Biology, 2000

The DNA polymerase of bacteriophage T4, product of phage gene 43 (gp43), has served as a model re... more The DNA polymerase of bacteriophage T4, product of phage gene 43 (gp43), has served as a model replicative DNA polymerase in nucleic acids research for nearly 40 years. The base-selection (polymerase, or Pol) and editing (3′-exonuclease, or Exo) functions of this multifunctional protein, which have counterparts in the replicatioe polymerases of other organisms, are primary determinants of the high fidelity of DNA synthesis in phage DNA replication. T4 gp43 is considered to be a member of the “B family” of DNA-dependent DNA polymerases (those resembling eukaryotic Pol α) because it exhibits striking similarities in primary structure to these enzymes. It has been extensively analyzed at the genetic, physiological, and biochemical levels; however, relationships between the in vivo properties of this enzyme and its physical structure have not always been easy to explain due to a paucity of structural data on the intact molecule. However, gp43 from phage RB69, a phylogenetic relative of ...