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J B Lingrel

Publications and source records attributed to J B Lingrel.

At least 145 records · Page 8Linked to original sources
PubMed1981

Transcription of the beta-like globin genes and pseudogenes of the goat in a cell-free system.

We have examined the transcription in a cell-free system of all the members of a highly regulated gene family, the beta-like hemoglobin genes of the goat. These five genes, which code for embryonic, fetal, juvenile, and adult beta-globin proteins, are all transcribed to roughly the same extent in the in vitro system. In all cases initiation of transcription is accurate. However, two goat beta-globin pseudogenes, as well as several artificially constructed deletion mutants, are not transcribed in vitro. A common feature of the transcriptionally inactive genes is the lack of an AT-rich consensus sequence just upstream of the presumptive initiation site.

Animals↗
PubMed1981

Two related pseudogenes are the result of a gene duplication in the goat beta-globin locus.

We report the nucleotide sequence of two goat beta-globin pseudogenes, psi beta X and psi beta Z, each of which is linked upstream from a functional goat beta-globin gene. Comparison to the functional beta A gene indicates that these two pseudogenes share several identical mutations. These include alterations of the ATA box, an early frameshift mutation in exon 1 and alterations of the universal GT/AG excision sequence of IVS1. The shared deleterious mutations indicate that the two pseudogenes diverged from a common defective sequence. Comparison of the pseudogenes with the functional beta A and beta C genes reveal that the psi beta X-beta C pair and psi beta Z-beta A pair may have been created by a single en bloc duplication event. The persistence and duplication of defective sequences may indicate a role for these pseudogenes as a spacer or regulator.

Animals↗
PubMed1981

Structure and evolution of goat gamma-, beta C- and beta A-globin genes: three developmentally regulated genes contain inserted elements.

We have determined the complete nucleotide sequence of the goat fetal (gamma), preadult (beta C) and adult (beta A) globin genes. In contrast to other globin genes expressed in different stages of development, these three genes are highly homologous (approximately 90%) in both their coding and noncoding regions. The only major difference between them results from elements inserted into their large introns: gamma contains a 247 bp insertion, beta A contains a 318 bp insertion, and beta C contains both a 252 bp insertion and a 60 bp deletion. Based on comparisons of these three genes to each other and to two goat globin pseudogenes, psi beta X and psi beta Z, we conclude that gamma, beta A and beta C have diverged relatively recently, and that the two gene pairs psi beta X - beta C and psi beta Z-beta A arose via a block duplication of an ancestral pseudogene-functional gene pair. Moreover, as opposed to the human fetal genes (G gamma and A gamma), the goat gamma gene appears to be a true beta-like gene that has fetal-specific function. The insertions in gamma, beta A and beta C contain sequence features characteristic of transposon-like elements, and appear to be represented in multiple copies throughout the genome.

Animals↗
PubMed1980

Identification by nucleotide sequence analysis of a goat pseudoglobin gene.

We have recently prepared a recombinant library of goat genomic DNA and have isolated clones containing th known goat globin genes. These include the alpha, gamma, beta C and beta A genes. In addition to these, another beta-like sequence has been observed. In this communication we report the complete nucleotide sequence of this gene, excluding a portion of the large intervening sequence. Several features suggest that this is a non-functional or pseudoglobin gene. The alterations include a frameshift mutation, substitution of the heme-binding histidines, a mutated termination codon, a change in the GT/AG excision sequence of the 5' end of the first intervening sequence, an AT rich sequence in the 3' untranslated region, and a mutated Hogness-Goldberg box. We conclude that this gene cannot function in the synthesis of globin.

Animals↗
PubMed1980

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library.

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.

Animals↗
PubMed1980

Unusual sequence homology at the 5-ends of the developmentally regulated beta A-, beta C-, and gamma-globin genes of the goat.

We have constructed a recombinant library of goat DNA and have isolated clones containing the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene. These genes are switched on and off during development and, thus, provide a model system for the study of gene regulation. To identify regions that may be involved in this switch, we determined the sequence of the areas surrounding the 5' ends of the beta A-, beta C- and gamma-globin genes. Surprisingly, the sequences of the beta A-, and beta C-globin genes are identical, beginning with the translation initiation codon and extending 131 base pairs (bp) in the 5' direction. At this point, two nucleotide differences are seen and only six additional differences are encountered when the sequences are extended 144 bp further in the 5' direction. Furthermore, the nucleotide sequence of the 5' end of the gamma-globin gene is very similar to those of the beta A-0, and beta C-globin genes. Only three nucleotide differences are found in the gamma-globin gene within the 131-bp region in which the beta A- and beta C-globin genes are identical. We conclude that this identical region cannot contain regulatory signals that mediate the switch from beta C to beta A expression.

Animals↗
PubMed1979

Synthesis and turnover of globin mRNA in murine erythroleukemia cells induced with hemin.

When murine erythroleukemia (MEL) cells are induced with hemin, they carry out several early functions of the erythroid program. However, they do not become committed to terminal differentiation nor do they become benzidine positive. This is in contrast to MEL cells induced with dimethyl sulfoxide (Me(2)SO) which undergo a more complete program of erythroid differentiation. In order to determine the relationship between commitment and various events in the erythroid program, we compared the induction of MEL cells with hemin and with Me(2)SO. The amount of globin mRNA accumulated in inducing MEL cells and the rate of its synthesis and turnover were quantitated. Although MEL cells induced with hemin accumulated significantly less globin mRNA than did cells induced with Me(2)SO, the rate of synthesis of globin mRNA was the same in fully induced cells, irrespective of inducer. Therefore, there is no evidence that induction with hemin produces an early program that is different or altered from that which is part of Me(2)SO induction. MEL cells induced with Me(2)SO specifically destabilize their globin mRNA after 4 days of induction. This raises the question of whether this destabilization of globin mRNA is an independently programmed late event, as suggested by the time of its occurrence, or, alternatively, whether it might be the inevitable consequence of an early event(s). For instance, destabilization might be linked to the synthesis or translation of globin mRNA. Because MEL cells induced with hemin do not destabilize their globin mRNA, we have concluded that this turnover of globin mRNA is a late event, occurring only in a committed cell population.

Animals↗
PubMed1978

A change in the stability of globin mRNA during the induction of murine erythroleukemia cells.

The stability of globin mRNA in murine erythroleukemia cells (Friend cells) before and during DMSO-induced differentiation was investigated. Cells were exposed to 3H-uridine for 2 hr and then transferred to medium without the radioactive precursor. The loss of radioactivity in total RNA, poly(A)-containing RNA and globin mRNA was followed. The globin mRNA was isolated using a highly specific globin cDNA column. In uninduced cells and cells early in differentiation, the globin mRNA decays with a half-life of less than 50 hr. After 4 days of induction, the globin mRNA decays with a half-life of 17 hr, demonstrating a change in stability during the induction process. Although the stability of globin mRNA changes during induction, this is not true for total poly(A)-containing RNA. At all times of induction, the poly(A)-containing RNA decays as two populations, one with a half-life of 6 hr and the other with a half-life of 36 hr. The half-life of the rRNA also remains unchanged during differentiation.

Cell Differentiation↗
PubMed1978

The location of the globin mRNA sequence within its 16S precursor.

The coding sequence of globin mRNA has been located at or very close to the 3' end of its poly(A)-containing 16S precursor. The 16S RNA was annealed to globin cDNA and the hybrid digested with ribonuclease H. The undigested fragment did not bind to oligo(dT)-cellulose and its size was that expected for the intact 5' portion of the precursor.

Animals↗
PubMed1978

Sequence organization of the beta-globin mRNA precursor.

The sequence organization of the beta-globin mRNA precursor has been determined directly by analyzing the resistant fragments from the RNase A digestion of the precursor RNA-globin cDNA hybrid. Three fragments are obtained which proves that the beta-globin mRNA sequence in its precursor is split into three discontinuous segments. The two intervening sequences in the beta-globin gene are therefore transcribed and removed during mRNA maturation.

Base Sequence↗
PubMed1977

Methylated nucleosides in globin mRNA from mouse nucleated erythroid cells.

Poly(A)-containing mRNAs labeled with [methyl-3H]methionine were isolated from nucleated erythroid cells obtained from the spleens of anemic mice. The RNAs were further separated into non-globin poly(A)-containing RNAs and highly purified globin mRNA by globin cDNA-cellulose affinity chromatography. DEAE-Sephadex column chromatography of the T2 ribonuclease digestion products of the cDNA-purified globin mRNA fraction yielded methylated resistant fragments with charges of -4.7 (Cap 1) and -5.3 (Cap 2). Digestion of the non-globin RNA fraction revealed a similar pattern with the addition of a methylated mononucleotide identified as 6-methyladenosine at -2 charges. Alkaline phosphatase treatment of the T2 resistant fragments reduced their charges by approximately 2, which is consistent with the removal of one terminal phosphate. Treatment of the globin T2 and alkaline phosphatase-resistant fragments withpenicillium P1 nuclease and alkaline phosphatase yielded a P1-resistant core structure in both fragments. In addition to the core, 2'-O-methylcytidine (Cm) was released from the more negatively charged globin fragment. The P1-resistant cores of the cap structures eluted from DEAE-Sephadex with the known standard m2G5'ppp5'Am and were found to be pyrophosphatase-sensitive establishing a 5'-5'-triphosphate linkage. The pyrophosphatase and alkaline phosphatase digestion products of the globin Cap 1 and Cap 2 core structures were analyzed by high voltage electrophoresis and paper chromatography and found to be 7-methyiguanosine (m7G) and the dimethylated nucleoside 6-methyl-2'-O-methyladenosine (N6mAm). A small amount of the singularly methylated adenosine, 2'-O-methyladenosine (Am) was also observed. The predominant sequences of the methylated nucleosides in the globin cap structures are therefore m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm.

Animals↗
PubMed1977

Purification of biologically active globin mRNA using cDNA-cellulose affinity chromatography.

A complementary DNA (cDNA) copy of mouse globin mRNA was synthesized using the RNA-dependent DNA polymerase from avian myeloblastosis virus and the oligo(dT) covalently attached to cellulose as primer. All four deoxyribonucleotide triphosphates, NaCl, the globin mRNA template, and an oligo(dT) primer were required for optimal synthesis of cDNA. By saturating the primer sites using a 3-fold excess of mRNA, sufficient concentrations of immobilized cDNA could be synthesized to allow the hybridization reactions to be performed using an excess of globin cDNA. Conditions which permitted the annealing of globin mRNA to cDNA-cellulose were selected and the sequence specificity for hybridization to cDNA-cellulose was determined using 28 S ribosomal RNA, polyadenylic acid, and mouse L-cell RNA. Both analytical and preparative applications of this chromatographic medium were explored. When radioactively labeled poly(A)-containing 9 S RNA isolated from nucleated erythroid cells was analyzed by affinity chromatography on globin cDNA-cellulose, 46 per cent of the applied radioactivity hybridized to the cDNA-cellulose column. Only 1 per cent of the labeled RNA was retained by the column during reapplication of the unbound fraction, while 96 per cent of the bound RNA reannealed to cDNA-cellulose. Hybridizations utilizing unfractionated RNA extracts from either mouse reticulocytes or nucleated erythroid cells provided a one-step purification method for globin mRNA sequences. The relative purity of the RNA isolated by cDNA-cellulose affinity chromatography was determined by hybridization kinetic analysis. The cDNA-bound fraction obtained from the unfractionated RNA of either cell type was shown to have a Crt1/2 of 2.7 x 10-3. This represents a 60-fold purification of the globin sequences present in reticulocyte polysomal RNA and a 280-fold enrichment of the globin mRNA in nucleated erythroid cells. Hybridization to cDNA-cellulose did not result in any change in the sedimentation rate of globin mRNA. Furthermore, experiments were performed which demonstrated that the globin mRNA isolated by hybridization to cDNA-cellulose retained its biological activity when assayed in a wheat germ cell-free lysate.

Animals↗
PubMed1977

Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells.

Nucleated erythroid cells were incubated for 10 min in the presence of [5-3H]uridine, and the total RNA was isolated by three different extraction procedures. RNA containing globin messenger RNA sequences was purified from other cellular RNAs by selective hybridization to globin complementary DNA cellulose. Depending upon the extraction procedure employed, 0.4-0.6% of the radioactively-labeled total cellular RNA applied to the column annealed to globin complementary DNA cellulose. The annealed RNA was treated with formaldehyde and analyzed by formaldehyde/polyacrylamide gel electrophoresis. Mature globin mRNA and an RNA migrating at approximately 15 S were observed. No globin mRNA containing sequences larger than 20 S were present. The 15S RNA was partially resolved from mature globin mRNA by neutral sucrose density gradient centrifugation. The RNA isolated from the heavy region of this gradient migrated as 15 S in the formaldehyde/polyacrylamide gels and retained its ability to quantitatively anneal to globin complementary DNA cellulose. On the basis of these observations, we conclude that nucleated erythroid cells obtained from the spleens of anemic mice have a 15S RNA which contains globin mRNA sequences. The 15S RNA is not an aggregate and is a good candidate for a globin mRNA precursor.

Animals↗
PubMed1976

Shortening of the poly(A) region of mouse globin messenger RNA.

Nucleated erythroid cells isolated from the spleens of anemic mice were used to investigate the processing of the polyadenylic acid region of globin mRNA. Cells were labeled in media containing [3H] adenosine and transferred to media containing no radioactive precursor and incubated further in the presence or absence of actinomycin D. After various times following the transfer of the cells, globin mRNA was isolated using a combination of oligo(dT)-cellulose affinity chromatography, sucrose density centrifugation, and globin cDNA (the complementary DNA copy of globin mRNA)-cellulose affinity chromatography. The size of the poly(A) region was determined by polyacrylamide gel electrophoresis of the T1 and pancreatic RNase-resistant fragments. The prelabeled poly(A) region which initially comprises approximately 150 adenylate residues was found to become shorter with time, both in cells incubated in medium containing no radioactive precursor and in the presence of actinomycin D. After 9 h of incubation in the presence of actinomycin D, two major size classes of poly(A) were observed, one containing 35 to 45 adenylic acid residues and the other containing 55 to 65 residues. These two size classes are similar to those found in circulating reticulocytes suggesting that the poly(A) shortening observed in these cell incubation studies is similar to that which occurs in vivo. Two protein synthesis inhibitors, emetine and cycloheximide, were investigated with respect to their effect on poly(A) shortening. Neither drug inhibited the shortening of the poly(A) region of globin mRNA, suggesting that protein synthesis is not required for this process to occur.

Animals↗
PubMed1976

Molecular weight determinations on the alpha-globin and beta-globin mRNAs.

The globin mRNAs containing between 30 and 40 polyadenylate residues can be separated from thos mRNAs containing longer poly(A) regions by Millipore filter binding. The molecular weights of the alpha-and beta-globin mRNAs containing this size class of poly(A) have beed determined by lectrophoresis on 3.7% polyacrylamide gels in the presence of 99% formamide. Because the number of adenylic acid residues in these mRNAs is known, the number of non-poly(A) nucleotides can be accurately calculated. The molecular weight of the beta-globin mRNA is 235 000 +/- 28 000 (736 +/- 88 nucleotides) and that of the alpha-globin mRNA is 208 900 +/- 43 870 (653 +/- 78 nucleotides). By subtracting the number of nucleotides in the coding and poly(A) regions, the number of non-coding nucleotides in the beta-globin mRNA were calculated to be 261, 69 more than the 193 present in the alpha-globin mRNA. Comparison of size estimates of newly synthesized globin mRNAs containing longer average lengths of poly(A) shhowed that there is no comparable processin of the 5' termini of the alpha-and beta-globin mRNAs concomitant with the stepwise degradation of the poly(A) regions which occur as the mRNAs mature.

Animals↗