Search PubMed⌕ Search

Biomedical subjects

E Goldman

Publications and source records attributed to E Goldman.

At least 37 records · Page 2Linked to original sources
PubMed1995

Consecutive low-usage leucine codons block translation only when near the 5' end of a message in Escherichia coli.

Insertion of nine consecutive low-usage CUA leucine codons after codon 13 of a 313-codon test mRNA strongly inhibited its translation without apparent effect on translation of other mRNAs containing CUA codons. In contrast, nine consecutive high-usage CUG leucine codons at the same position had no apparent effect, and neither low- nor high-usage codons affected translation when inserted after codon 223 or 307. Additional experiments indicated that the strong positional effect of the low-usage codons could not be accounted for by differences in stability of the mRNAs or in stringency of selection of the correct tRNA. The positional effect could be explained if translation complexes are less stable near the beginning of a message: slow translation through low-usage codons early in the message may allow most translation complexes to dissociate before they read through.

Blotting, Northern↗
PubMed1994

Clustering of low usage codons and ribosome movement.

A model is presented in which the distribution of low-usage codons in a message is a major factor in determining the impact that they will have on the translation rate and distribution of ribosomes on that message. This model is based on the assumption that low-usage codons are translated more slowly than normal codons, an assumption supported by various lines of published experimental evidence. Although the parameters used to develop this model are somewhat arbitrary, the main conclusions of this paper are consistent with a wide variation in the values of those parameters. In the model, low-usage codons arranged in clusters are much more effective in blocking ribosome movement on the message than ones that are dispersed. The effective size of the cluster is limited to the dimensions of the ribosome. It has been estimated that ribosomes on a message are spaced at least 27 nucleotides or nine codons apart. A ribosome translating a cluster of nine codons in which some or all of the codons are low-usage will move more slowly than over a comparable stretch of message containing no low-usage codons. Owing to ribosome size, the ribosome immediately behind the stalled ribosome will move as slowly; it must wait for the stalled ribosome to move on before it can even begin to translate the difficult region containing the low-usage codons. When the low-usage codon cluster is at the 3' end, the message will eventually be occupied by a ribosome jam that will transmit back to the 5' end of the message. In the steady state, the slowing effect imposed by a cluster of nine low-usage codons at the 3' end of a message would be just as great as if the entire message was composed of them. If the cluster is situated in the middle of a message, the ribosomes will form a jam upstream of the cluster. The ribosome density downstream of the cluster will be considerably reduced from what it would be for the same message with no cluster. If the cluster is at the 5' end of the message, the density of ribosomes will be reduced over the entire length of the message but the overall translation rate per ribosome will be only slightly reduced. However, owing to the reduced number of ribosomes initiating, the efficiency of the message in protein synthesis will be considerably reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Analysis of intron 22 inversions of the factor VIII gene in severe hemophilia A: implications for genetic counseling.

Intrachromosomal recombinations involving F8A, in intron 22 of the factor VIII gene, and one of two homologous regions 500 kb 5' of the factor VIII gene result in large inversions of DNA at the tip of the X chromosome. The gene is disrupted, causing severe hemophilia A. Two inversions are possible, distal and proximal, depending on which homologous region is involved in the recombination event. A simple Southern blotting technique was used to identify patients and carriers of these inversions. In a group of 85 severe hemophilia A patients, 47% had an inversion, of which 80% were of the distal type. There was no association with restriction fragment length polymorphism (RFLP) haplotypes. The technique has identified a definitive genetic marker in families previously uninformative on RFLP analysis and provided valuable information for genetic counselling information may now be provided for carriers without the need to study intervening family members and the diagnosis of severe hemophilia A made in families with only a nonspecific history of bleeding. Analysis of intron 22 inversion should now be the first-line test for carrier diagnosis and genetic counselling for severe hemophilia A and may be particularly useful when there is no affected male family member or when intervening family members are unavailable for testing.

Chromosome Inversion↗
PubMed1994

Role of carboxy-terminal region in proofreading function of methionyl-tRNA synthetase in Escherichia coli.

The synthetic and editing functions of three forms of Escherichia coli methionyl-tRNA synthetase with different C-terminal sequences have been compared in vivo and in vitro. These forms include a full-length wild-type dimer (MRS676), a truncated monomer (MRS547) believed to be equivalent to the biologically active large tryptic fragment, and a third form denoted MRS581*. DNA sequencing revealed that MRS581* is predicted to contain 18 additional amino acids from the wild-type full-length sequence at the carboxy terminus of truncated form MRS547, and this is then fused to an additional 16 amino acids encoded by vector pBR322. Both MRS676 and MRS581* were found to edit endogenous homocysteine about 20-fold more efficiently than MRS547 in vivo. However, the three methionyl-tRNA synthetases edited exogenously supplied homocysteine in bacterial cultures to similar extents. Purified proteins exhibited no significant differences in editing function in vitro. Synthetic activity of purified MRS676 in vitro was found to be about 2.5-fold higher per subunit compared to the shorter forms of the enzyme. The C-terminal region in E. coli methionyl-tRNA synthetase is thus suggested to play an important role in editing in vivo, most likely by allowing interaction of the enzyme with the methionine biosynthetic pathway. These data support a model of channeling of at least some metabolites in bacterial protein synthesis.

Escherichia coli↗
PubMed1993

Increased ribosomal accuracy increases a programmed translational frameshift in Escherichia coli.

We have tested the effect of increased ribosomal fidelity on a modified version of the programmed release factor 2 (RF2) translational frameshift. In the constructs tested, the original UGA codon at the site of the shift was replaced by either of two sense codons, UGG (tryptophan), which allows a frameshift of approximately 13%, or CUG (leucine), which allows a frameshift of only approximately 2%. We confirmed the results of Curran and Yarus [Curran, J. F. & Yarus, M. (1989) J. Mol. Biol. 209, 65-77] in a wild-type ribosomal host, including a reduction of the UGG shift following induction of tRNA(Trp) from a plasmid copy of the tRNA gene. But to our surprise, in a hyperaccurate streptomycin pseudo-dependent host, the UGG frameshift increased to more than 50%. When we added a tRNA(Trp) plasmid to these cells, induction of the tRNA(Trp) gene reduced the shift back to approximately 7%. Messenger RNA levels did not vary greatly under these different induced conditions. Other increased accuracy alleles also showed increased frameshifting with UGG at the frameshift site. All increased accuracy alleles led to slower translation rates, and there appeared to be a proportionality between the extent of reduction of synthesis for the in-frame reporter and the extent of UGG frameshift for the out-of-frame reporter. There were little effects of increased accuracy on the lower level CUG frameshift. However, over-production of the cognate tRNA(1Leu) dramatically reduced even this lower level of shift, despite the fact that tRNA(1Leu) is already the most abundant isoacceptor in Escherichia coli. These results can be rationalized by following the hypothesis of Curran and Yarus as follows: with wild-type ribosomes, limited availability of tRNA(Trp) (about 1% of total tRNA) facilitates a pause at the UGG codon (due to the vacant A site), allowing increased opportunity for ribosome realignment. Excess tRNA(Trp) reduces the time the A site is vacant and thus reduces the frameshift. The slower hyperaccurate ribosomes increase the pause time and thus increase the opportunity for shifting, a process again reversed by increasing the in-frame cognate tRNA(Trp). These data provide strong support for a model in which the extent of ribosome pause time at a programmed frameshift site is a major determinant in the efficiency of the frameshift and in which tRNA availability can be a major influence on this process.

Base Sequence↗
PubMed1993

Synthesis of homocysteine thiolactone by methionyl-tRNA synthetase in cultured mammalian cells.

Homocysteine thiolactone is a product of an error-editing reaction, catalyzed by Escherichia coli and Saccharomyces cerevisiae methionyl-tRNA synthetases, which prevents incorporation of homocysteine into tRNA and protein both in vitro and in vivo. Here, homocysteine thiolactone is also shown to be synthesized by cultured mammalian cells such as human cervical carcinoma (HeLa), mouse renal adenocarcinoma (RAG), and Chinese hamster ovary (CHO) cells labeled with [35S]methionine, but not by normal human and mouse (Balb/c 3T3) fibroblasts. A temperature-sensitive methionyl-tRNA synthetase mutant of CHO cells, Met-1, does not make the thiolactone at the non-permissive temperature. The data indicate that methionyl-tRNA synthase is involved in synthesis of homocysteine thiolactone in CHO cells, thereby extending this important proofreading mechanism to mammalian cells.

3T3 Cells↗
PubMed1993

Haemophilia B caused by a missense mutation in the prepeptide sequence of factor IX.

In the course of analysing mutation in the factor IX gene from 200 haemophilia B patients in Sweden and the UK, we have identified one patient with a prepeptide missense mutation. He has severe, antigen negative haemophilia, and complete analysis of his coding sequence reveals a single base transversion (A-->T) causing substitution of isoleucine by asparagine at position -30. This change disrupts the hydrophobic core of the prepeptide, a feature which is required for secretion. Thus, haemophilia in this patient is caused by a failure to secrete factor IX from the hepatocytes.

Amino Acid Sequence↗
PubMed1993

A family with HIV and haemophilia.

An Indian Sikh family affected with both HIV and haemophilia is described. The use of interpreters; the regular follow-up in the Concorde trial; family therapy and help with practical problems have been approaches used to help the family despite language and cultural differences.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Methionine-mediated lethality in yeast cells at elevated temperature.

Saccharomyces cerevisiae cells grown at 30 degrees C in minimal medium containing methionine lose viability upon transfer to 45 degrees C, whereas cells grown in the absence of methionine survive. Cellular levels of two intermediates in the sulfate assimilation pathway, adenosine 5'-phosphosulfate (APS) and adenosine 5'-phosphosulfate 3'-phosphate, are increased by a posttranslational mechanism after sudden elevation of temperature in yeast cultures grown in the absence of methionine. Yeast cells unable to synthesize APS because of repression by methionine or mutation of the MET3 gene do not survive the temperature shift. Thus, methionine-mediated lethality at elevated temperature is linked to the inability to synthesize APS. The results demonstrate that APS plays an important role in thermotolerance.

Adenine Nucleotides↗
PubMed1993

Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system.

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages. Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNA(AGG) in the cell to become sequestered in translating peptidyl-tRNA(AGG) -mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome of frameshift, hop, or terminate translation.

Arginine↗
PubMed1993

Children of HIV positive haemophilic men.

All 14 children who were conceived at the time their 12 haemophilic fathers were anti-HIV positive were shown to be HIV negative and to be healthy physically and mentally. Eleven of the 12 female partners have remained anti-HIV negative and one has seroconverted. Despite counselling it is likely that children will continue to be conceived by anti-HIV positive haemophiliacs.

Adult↗
PubMed1992

Probing the primary donor environment in the histidineM200-->leucine and histidineL173-->leucine heterodimer mutants of Rhodobacter capsulatus by light-induced Fourier transform infrared difference spectroscopy.

Light-induced P+QB-/PQB FTIR difference spectra of reaction centers (RCs) have been obtained from chromatophores lacking light-harvesting B800-850 antenna for Rhodobacter capsulatus wild type (WT) and for the two mutants HisM200-->Leu and HisL173-->Leu. The primary donor (P) in both mutants consists of a bacteriochlorophyll-bacteriopheophytin heterodimer. The most prominent difference between the WT and the mutant spectra is in the 1600-1200-cm-1 region. The WT spectrum displays large positive bands at approximately 1290, 1500-1430, and 1580-1530 cm-1. These three bands are either small or altogether absent in the heterodimer spectra. In addition, both heterodimer spectra compare well with the electrochemically generated BChla+/BChla spectrum [Mäntele, W.G., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472]. These observations indicate that the positive charge is localized on the monomeric BChl in the heterodimers. The overall shape of the ester and keto C = O signals in the BChla+/BChla spectrum is maintained in the in situ spectra although significant differences are observed in the frequency, width, and splitting of the bands. The shape of the signal at 1757/1744 cm-1 in HisL173-->Leu is comparable to the 1751/1737-cm-1 signal of BChla+/BChla in tetrahydrofuran, indicating a free 10a ester C = O of PM in HisL173-->Leu. The reduced amplitude of the negative 1740-cm-1 feature in both HisM200-->Leu and WT spectra suggests a hydrogen-bonded 10a ester C = O for PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriochlorophylls↗